CLEANROOM

CONTAMINATION
PREVENTION & CONTROL

A PRACTICAL GUIDE
TO THE SCIENCE

Ziva Abraham
Morgan Polen
Editors
 Cleanroom
Contamination
Prevention & Control

A Practical Guide
To the Science

Ziva Abraham
Morgan Polen
Editors

PDA
Bethesda, MD, USA

DHI Publishing, LLC

River Grove, IL, USA
10 9 8 7 6 5 4 3 2 1

ISBN: 978-1-942911-54-8
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 CONTENTS

PREFACE.......................................................................................................xi

ABOUT THE AUTHORS.........................................................................xiii

INTRODUCTION................................................................................... xvii

1 CONTAMINATION RELATED CASE STUDIES

AND 483 OBSERVATIONS................................................................ 1
Morgan Polen and Ziva Abraham
Cost of Contamination............................................................................................................. 1
Primary Causes of Contamination Events....................................................................... 2
Particulate Contamination...................................................................................................... 3
Risk to Patient from Particulates.......................................................................................... 5
Static Contamination................................................................................................................. 8
Microbial Contamination......................................................................................................... 8
Safeguarding Against Contamination.................................................................................. 9
Case Study 1 Particulate Contamination and Electrostatic
Charge in Medical Device Manufacturing..............................................................10
Case Study 2 Cleanroom Design for Manual Aseptic Process
– 483 Observation...........................................................................................................12
Case Study 3 Cleanroom and Equipment Design, Open Door
Interventions – 483 Observation..............................................................................14
Case Study 4 Liquid Filling Line – 483 Observation..................................................15
Case Study 5 Terminally Sterilized Filling Operation with
Mold Contamination – 483 Observation..............................................................18

iii
iv Cleanroom Contamination Prevention and Control
Case Study 6 Barrier System Design Flaws and Cleanroom
Integration Mistakes........................................................................................................21
Case Study 7 Airflow Velocity – 483 Observation....................................................22
Case Study 8 Importance of Ergonomics in Aseptic
Processing – 483 Observation....................................................................................23
Case Study 9 Lack of Scientific Rationale in Environmental Monitoring
– 483 Observation...........................................................................................................25
Case Study 10 Non-Integral Cleanroom Materials – 483 Observation............27
Case Study 11 Misuse of Disinfectants............................................................................28
Case Study 12 Disinfectants Misuse.................................................................................30
Case Study 13 Nontuberculous Mycobacteria in Pharmaceutical
Water System....................................................................................................................31
Case Study 14 Monitoring Equipment.............................................................................33
Case Study 15 HMI Exhaust Fan as Source of Biofilm Formation
in Manufacturing Equipment........................................................................................33
Case Study 16 Manufacturing Equipment Cleaning Validation
and Product Testing Failures........................................................................................34
Case Study 17 Contamination in an ISO Class 8 Purification Room.................36
Case Study 18 Product Contamination..........................................................................37
Case Study 19 Booklice in Stability Chamber..............................................................38
Case Study 20 Contamination of Incubators................................................................39
Case Study 21 Laboratory Related Contamination...................................................40
Case Study 22 Microbiology Media...................................................................................41
Conclusion...................................................................................................................................43
References....................................................................................................................................43
About the Authors...................................................................................................................45

2 PAPER COMPLIANCE VS FACTUAL

CONTAMINATION CONTROL..................................................... 49
Ziva Abraham...............................................................................................................................49
Contamination Risk..................................................................................................................51
Managing Risk..............................................................................................................................69
Conclusion...................................................................................................................................72
References....................................................................................................................................74
About the Author.....................................................................................................................76
 Contents v

3 DESIGN BASICS OF PHARMACEUTICAL

CLEANROOMS................................................................................... 79
Wei Sun..........................................................................................................................................79
Design Considerations for Pharmaceutical Cleanrooms.........................................80
Piping and Instrumentation Diagrams..............................................................................81
Particle Sources and Control Methods...........................................................................84
ISO Class and GMP Grade Determination ..................................................................84
Contamination Controls in Room Airflow Pattern Design....................................85
Airflow Intensity Determination........................................................................................87
HVAC System Configurations.............................................................................................88
Energy Saving Measures to Lower Cleanroom Air Change Rates.......................90
Cleanroom Pressure and Control Strategies................................................................93
Multiple-Rooms (Suite) Under Multistage Pressurizations......................................96
Temperature and Relative Humidity.................................................................................97
Noise Control.............................................................................................................................98
Environmental Monitoring and Controls........................................................................99
Airlocks.......................................................................................................................................100
Barrier Technology................................................................................................................102
Isolators......................................................................................................................................103
Restricted Access Barrier Systems.................................................................................105
Gloveboxes ..............................................................................................................................107
Laminar Air Flow Units.......................................................................................................108
Surrounding Cleanroom......................................................................................................109
Environmental Control........................................................................................................109
Decontamination....................................................................................................................110
Maintainability..........................................................................................................................111
Controls, Monitors, and Alarms......................................................................................112
References.................................................................................................................................114
About the Author..................................................................................................................114

4 CLEANROOM DESIGN QUALITY CONTROL

AND DOCUMENTS REVIEW.......................................................117
Wei Sun
User Requirements Specification for Design Documents....................................118
Progressive Flow of Design Phases.................................................................................119
Design Quality Control Plan on Design Documents ............................................122
Design Reviews.......................................................................................................................123
References.................................................................................................................................125
About the Author..................................................................................................................126
vi Cleanroom Contamination Prevention and Control

5 CLEANROOM AIRFLOW VALIDATION

IN DESIGN PHASE..........................................................................127
Wei Sun
Factors Impacting Cleanroom..........................................................................................128
Airflow Pattern and Resulting...........................................................................................128
ISO Class Air Cleanliness....................................................................................................128
How to Assure Required Outcomes during Design Phase ................................128
Airflow Verification Requirements by ISO Standard
and Design Guidelines ................................................................................................129
CFD Principle .........................................................................................................................131
CFD Practices for Cleanrooms ......................................................................................131
Applications of CFD Simulation and Analysis for Cleanrooms..........................133
Examples of CFD Modeling, Simulation and Analysis............................................136
References.................................................................................................................................142
About the Author..................................................................................................................143

6 AIR FLOW VISUALIZATION: THE MOST

MISUNDERSTOOD AND UNDERUTILIZED
CONTAMINATION CONTROL TOOL......................................145
Morgan Polen
Air Flow Visualization for Pharmaceutical and
Other Medical Product Cleanrooms....................................................................145
What is Air Flow Visualization (AKA Smoke Studies)?..........................................147
Air Flow Visualization is a Tool for Contamination Control...............................148
Types of Air Flow in Cleanrooms...................................................................................150
Why Perform Air Flow Visualization Studies?...........................................................159
What are the Benefits from Air Flow Visualization?...............................................161
What are the Consequences of Improperly..............................................................163
Conducted Air Flow Visualization Studies?.................................................................163
What are the International Standards? ........................................................................166
What are the Regulatory Requirements,.....................................................................168
Guidelines or Expectations?..............................................................................................168
Regulators Comments on Smoke Studies...................................................................176
Air Flow Visualization Applications................................................................................177
Types of Air Flow Visualization.........................................................................................182
Simulations of Activities During Air Flow Visualization.........................................185
Preparation for Dynamic Smoke Studies.....................................................................185
Fire and Smoke Detection Systems...............................................................................186
Tracer Particle Manifold Position.....................................................................................187
 Contents vii
Tracer Particles........................................................................................................................189
Conclusion................................................................................................................................193
References.................................................................................................................................193
About the Author..................................................................................................................195

7 STATIC CHARGE: THE UNSEEN CONTAMINANT...............197

Larry Levit
Undesirable Effects of Static Electricity........................................................................197
Underlying Science.................................................................................................................202
Cleanroom Electrostatic Management.........................................................................206
Summary....................................................................................................................................211
References.................................................................................................................................216
About the Author..................................................................................................................217

8 DESIGNING, COMMISSIONING AND RISK

ASSESSING CLEANROOMS FOR BIOCONTAMINATION
CONTROL.........................................................................................219
Tim Sandle
Introduction..............................................................................................................................219
Cleanrooms and Clean Air Devices...............................................................................221
Cleanrooms Design...............................................................................................................227
Commissioning a New Cleanroom................................................................................233
Commissioning (Qualifying) Cleanrooms....................................................................236
Microbial Control...................................................................................................................239
Tests Required for the Commissioning and Qualification
of Cleanrooms................................................................................................................247
Components Verification....................................................................................................247
Environmental Monitoring..................................................................................................270
Risk Based Approach to Cleanroom Assessments.................................................271
Summary....................................................................................................................................277
References.................................................................................................................................279
About the Author..................................................................................................................285
viii Cleanroom Contamination Prevention and Control

9 CLEANROOM COMMISSIONING AND

QUALIFICATION.............................................................................287
Wei Sun
Commissioning supervision...............................................................................................288
Qualifications ..........................................................................................................................291
Design Qualification..............................................................................................................293
Installation Qualification......................................................................................................294
Operational Qualification...................................................................................................296
Performance Qualification.................................................................................................298
Requalification..........................................................................................................................299
References.................................................................................................................................299
About the Author..................................................................................................................300

10 HUMAN BORNE CONTAMINATION –

WHEN CLEANROOM REUSABLE OR DISPOSABLE
GARMENTS CANNOT BE A GOOD BARRIER........................303
Jan Eudy
The Importance of Correct Selection of Reusable
or Disposable, Single-Use Cleanroom Garments...........................................304
The Cleanroom Laundering Process.............................................................................311
Sterile Cleanroom Garments ..........................................................................................313
Gamma Subcontractor Qualification ..........................................................................316
Routine Monitoring of Gamma Radiation...................................................................316
Certificate of Sterility...........................................................................................................317
Applications Specifically for Disposable........................................................................319
Single-Use Cleanroom Garments ..................................................................................319
References.................................................................................................................................323
About the Author..................................................................................................................327

11 VIABLE AND NON-VIABLE AIRBORNE PARTICLE

MONITORING EQUIPMENT SELECTION................................329
Bill Shade
Overview...................................................................................................................................329
Industry Standards and Guidelines.................................................................................331
For Non-Viable Particle Monitoring...............................................................................331
Airborne Particle Counter Technology and Specifications..................................340
Optical Particle Counters...................................................................................................346
Types and Applications........................................................................................................346
 Contents ix
Industry Standards, Guidelines and Practices
for Viable Particle Monitoring..................................................................................352
Conclusion................................................................................................................................362
References.................................................................................................................................363
About the Author..................................................................................................................365

12 FOOT AND WHEEL BORNE CONTAMINATION..................367

Krishna Bell and Ziva Abraham
Temporary and Permanent Sticky Mats.......................................................................370
References.................................................................................................................................379
About the Authors................................................................................................................381

13 VALIDATION OF DISINFECTANTS............................................383
Tim Sandle, Laura Guardi, Rachel Kirkham and Kim Morwood
Introduction..............................................................................................................................383
Defining a Disinfectant Validation Strategy and Selection
of Test Parameters........................................................................................................385
Use-Dilution and Suspension Tests................................................................................386
Disinfectant Surface Challenge Tests.............................................................................386
Modification of Test Parameters......................................................................................388
Validation of the Neutralizing Effect of Contact Plates.........................................399
Qualification of Hold Times for Diluted Disinfectants..........................................400
Field Trials..................................................................................................................................400
Re-validation.............................................................................................................................401
Considerations for Qualification of Hand Hygiene Products.............................401
Summary of Common Test Methods............................................................................402
Test Methods from CEN....................................................................................................402
AOAC Test Methods............................................................................................................407
Summary....................................................................................................................................410
References.................................................................................................................................411
About the Authors................................................................................................................415

14 CASE STUDIES FOR IMPROVING A RISK-BASED

CLEANING AND DISINFECTION PROGRAM.........................417
Jim Polarine and Beth Kroeger
Making the Dilution...............................................................................................................417
Use of the Disinfectant Use-Dilution in Cleanroom
Bucket Assemblies........................................................................................................418
x Cleanroom Contamination Prevention and Control
Application Method...............................................................................................................419
Changing out the Use-Dilution........................................................................................422
Cleaning versus Disinfection..............................................................................................423
Rotation......................................................................................................................................423
Controlling Residues.............................................................................................................425
Conclusion................................................................................................................................428
References.................................................................................................................................429
About the Authors................................................................................................................430

15 CLEANROOM CLEANING – CHOOSING, USING,

AND HANDLING CLEANING MATERIAL................................433
Matts Ramstorp
Introduction..............................................................................................................................433
Why is Cleaning of Cleanrooms and Clean Zones Critical?...............................434
Everything in a Cleanroom is Interconnected ..........................................................435
Visibility is a Major Problem During Cleaning and Disinfection.........................437
Cleaning Is Often The Most Contaminating Process in a Cleanroom............438
How to Minimize Dispersion of Particles During Cleaning.................................439
A Quick and Helpful Way to Determine if a Cleaning
Technique is Suitable for Cleanroom Use..........................................................440
Choice of Cleaning Material..............................................................................................440
Time Allowed for Cleaning................................................................................................441
How to Clean..........................................................................................................................442
Cleaning Operator Training...............................................................................................443
Cleaning Operator Behavior.............................................................................................443
Cleaning materials – Fundamental Properties...........................................................445
Cleanroom wipes...................................................................................................................446
Creating a Cleaning Program............................................................................................449
Cleaning of Cleanrooms from a Practical Approach..............................................451
Summary....................................................................................................................................457
References.................................................................................................................................457
About the Author..................................................................................................................458

INDEX.......................................................................................................459
 PREFACE

As the Head of Operations at Microrite, the repeat requests

for investigating similar contamination issues, warning letter
remediation and product failure issues made me recognize that
all these scenarios have one thing in common. The commonality
of these requests eluded to a basic knowledge gap in holistically
addressing contamination control. Though the current regulatory
thinking expects the evaluation of contamination control from a
holistic perspective; the deficiency in understanding the sources
of contamination and their relationship to subsequent steps in the
manufacturing process was lacking.

This book is strategically arranged for evaluating contamination

control holistically. As contamination control should be looked at
from design, manufacturing and an operations perspective, subject
matter experts, not generalists, in each area were approached to
contribute to this valuable compilation of the following science
based contamination control journey.

xi
xii Cleanroom Contamination Prevention and Control

The information in this book, which may be new to many

readers will help ignite curiosity and ultimately understanding
of what the sources of contamination can be and how to address
contamination prevention, control and monitoring. The purpose of
this book has been to create awareness and provide solutions. This
book will become a contamination control audit if read, understood
and applied.

Amir Abraham
Vice President
Microrite. Inc.
 ABOUT THE AUTHORS

Morgan Polen is a subject matter expert in the field of contamination

control, airflow visualization and particle monitoring in cleanrooms.
Morgan has over 35 years of industry experience working in
cleanrooms in all industries.

Morgan’s Technical Leadership:

• Member of International Disk Drive and Equipment Materials
Association (IDEMA) Standards Committee.
• Member of US Technical Advisory Group to ISO Technical
Committee 209 (international cleanroom standards i.e., ISO
14644 Cleanrooms and Associated Controlled Environments).
• Member of ASTM Subcommittee E55.06 Working Group “Guide
for Critical Airflow Visualization”.
• Board member of the Institute of Environmental Sciences and
Technology (IEST).

xiii
xiv Cleanroom Contamination Prevention and Control

Morgan has been instrumental in drafting and editing international

cleanroom standards, contamination control guidelines and best
practices. He has extensive experience working on cleanroom
projects in the United States, Canada, Mexico, Germany, Malaysia,
Taiwan, South Korea, Singapore, Thailand, United Kingdom, Ireland,
China, Philippines, India and Turkey and is a valuable resource in
addressing contamination control in critical environments for the
electronics, aerospace and healthcare industries.

As a key member of Microrite’s Expert Contamination

Control team, Morgan is instrumental in development of proactive
contamination control strategies through pragmatic risk assessment,
particle testing and airflow visualization. Morgan’s broad experience
troubleshooting contamination issues has helped companies with
FDA 483/warning letter remediation.

Ziva Abraham has over 35 years of academic, research, clinical and

industrial experience in Microbiology, and Quality Assurance. Ziva
has received her master’s degree in Microbiology and has conducted
research on developing Microbial Insecticides using entomogenous
bacteria and fungi towards her Ph.D. degree.

She worked as a clinical scientist for many years and established

clinical laboratory systems in Israel. In this capacity Ziva evaluated
the first automated microbial identification system, introduced
new technologies for HIV testing, and many rapid testing methods
into the group of laboratories she helped establish and manage
for Maccabi Medical. These laboratories conducted clinical
testing in microbiology, chemistry, parasitology, hematology and
immunology.

She is a passionate microbiologist and mycologist and provides

training worldwide on various microbiology and contamination
control topics. Her clinical experience helps her evaluate and
teach the clinical implications of microbial contamination on
patients. Ziva is passionate about fungi; as in her research she had
 About the Authors xv

collected, identified, and tested hundreds of species. She routinely

teaches hands-on fungal identification as well as investigation
and remediation of mold contamination and most importantly the
emergence of new fungal infections.

Ziva established Microrite, a California based consulting firm,

in 1998 as an independent microbiology consultant. Ziva used her
extensive research experience with fungi and clinical organisms
and their impact on human health to build an internationally
renowned contamination control team of experts. Her team consists
of facilities, cleanroom, airflow, static charge, validation, quality and
microbiology professionals with practical knowledge in their field
and involvement in industry standards and guidance organizations.
Ziva’s team works congruently to resolve client issues, prevent
contamination through design and help clients produce safe
products for human and veterinary use.
 INTRODUCTION

THE GOAL
The goal of this book is to impart knowledge about the various
aspects of contamination control as they relate to medical product
manufacturing and ultimately patient safety. A robust contamination
control plan means more than removing particulate or microbial
contamination from cleanroom surfaces and equipment; it starts at
the facility design phase. A well-designed facility, which is capable
of providing adequate air quality in all cleanrooms and protection of
open product in critical processing areas is the first step to preventing
contamination.

Subsequent to a good design are factors such as material flows,

personnel flows and waste flows that prevent ingress of con­
tamination or cross contamination. Removal and monitoring of
con­tamination are equally critical steps to managing contaminat­
ion control within a facility. To achieve this contamination control
goal across various aspects requires input from many disciplines,
experience, and knowledge base. This diversity and magnitude of
knowledge and experience is required to have a meaningful impact
on product quality. No sole resource can provide a comprehensive
solution to every element integral to the complex, highly technical,
and critical exercise of proactively mitigating contamination.

xvii
xviii Cleanroom Contamination Prevention and Control

In the past few years there has been a significant increase in the
number of warning letters and FDA 483 observations referring to smoke
studies, cleanroom design, ergonomics, data integrity in monitoring
and sterility failures. These observations, related to contamination,
denote multiple failures that are repetitive in nature including facility
design flaws, inadequate integration of barrier systems into the
cleanrooms, inadequate cleanroom qualification, poorly performed
smoke studies, inadequate cleaning and disin­fection, personnel and
material flows that are not conducive to contamination control, and
inefficiency of monitoring of contam­ination in cleanrooms.

Beyond aging facilities that tend to face more contamination

issues due to outdated designs, the newer facilities and barrier
system designs are not optimized from a holistic contamination
control effect perspective. This common oversight has led to
increased regulatory scrutiny even with newer facilities.

THE REASON
Review of warning letters, FDA 483 observations and data integrity
related issues combined with experience in the field of contamination
control revealed a repetitive pattern common across companies.
Facility design, barrier and cleanroom integration issues led to bad
airflows and ergonomics; these issues led to falsification of environ­
mental monitoring data and flawed aseptic practices. Ingress of
contamination due to inadequate personnel and material flows
combined with deficient cleaning and disinfection, and incorrect
supplies added to contamination load in critical areas. Additionally,
facilities, though validated, could not meet room requirements for
particle and microbial limits. Often companies did not sufficiently
address the root causes of recurring deficiencies, leading to repeat
observations by regulators.

These misunderstood aspects of contamination control and

lack of knowledge base prompted the development of this practical
guide using subject matter experts in each of the areas which impact
the quality of medicinal products.
 Introduction xix

THE READERS
This book has been developed to address the above-mentioned gaps
and provide the readers with an understanding of the common
errors made and guidance on the precautionary steps to be taken to
avoid contamination, harm to patient and regulatory scrutiny.

This book is written for professionals in every department

concerned with protecting the product from contamination. This
includes facilities, validation, manufacturing, quality assurance,
quality control, and QC microbiology. This book can greatly help
those who wish to expand their knowledge regarding specific steps
to take to prevent or mitigate contamination. It is also meant for those
who have expertise in one discipline and wish to better understand
the other disciplines as they relate to contamination control.

THE STRATEGY
Reading this book from beginning to end in a chronological order
can be utilized to develop a tool for performing a comprehensive
contamination control audit. Alternatively, as each chapter is a
lesson in a particular aspect of contamination control, the reader
may choose the chapters of interest.

This book contains 15 chapters in total starting with

Contamination Related Case Studies and 483 Observations (Chapter
1). Chapter 1 is a compilation of common mistakes made with an
explanation on how those mistakes could have been prevented.
These case studies set the tone to discuss shortcomings in detail in
subsequent chapters.

Paper Compliance vs Factual Contamination Control (Chapter

2) highlights the futility of performing a paper exercise of assigning
risk levels and scaling without having the knowledge base and
experience in assessing true risk. Pharmaceutical Cleanroom Design
(Chapter 3) addresses cleanroom design criteria that are conducive
to contamination control while Cleanroom Design Quality Control
xx Cleanroom Contamination Prevention and Control

and Documents Review (Chapter 4) emphasizes the importance of

independent review at various steps of cleanroom design to avoid
making mistakes that may lead to dire consequences. Cleanroom
Airflow Validation in Design Phase (Chapter 5) addresses com­
putational fluid dynamics (CFD) which is a new concept in
pharmaceutical manufacturing for preemptively ensuring adequate
airflows, particle generation and other control parameters at the
final cleanroom design phase before build.

Air Flow Visualization: the Most Misunderstood and

Underutilized Contamination Control Tool (Chapter 6) delves deep
into the common errors during smoke studies that have either led to
contamination of product, environmental monitoring, media fill or
product failures, and most importantly have led to increased scrutiny
by regulators over the past few years. Static Charge: The Unseen
Contaminant (Chapter 7) discusses the challenges of particulate and
microbial contamination due to static charge, a concept that should
be well understood as the industry moves towards automation for
manufacturing of medicinal products.

Designing, Commissioning and Risk Assessing Cleanrooms for

Biocontamination Control (Chapter 8) and Cleanroom Commission­
ing and Qualification (Chapter 9) discuss the steps for commissioning
and qualification required per industry standards and how to use
the information gathered during these activities to assess risk of
contamination.

Human Borne Contamination – When Cleanroom Reusable

or Disposable Garments Cannot be a Good Barrier (Chapter 10)
discusses gown choice, management and gowning practices. Often
gown choice and management gaps are a cause of human borne
contaminants causing failures which occur during aseptic practices,
environmental monitoring, media fills or sterility testing.

There are many criteria for choosing environmental monitoring

equipment; failure in understating these requirements leads to false
negative or false positive monitoring data. These requirements
 Introduction xxi

are clearly defined in Viable and Non-viable Particle Monitoring

Equipment Selection (Chapter 11).

Foot and Wheel Borne Contamination (Chapter 12) addresses

the routes of entry for contaminants into the cleanroom and how
to effectively stop this ingress. Cleaning and disinfection as well as
choice of cleaning supplies is a critical part of reduction and removal
of contamination. Disinfectant qualification is a subjective test, if not
performed well the data may be inconclusive and the disinfection
program may be ineffective. The requirements and test procedures
are clearly outlined and explained in Validation of Disinfectants
(Chapter 13).

The examples in Case Studies for Improving a Risk-Based

Cleaning and Disinfection Program (Chapter 14) highlight the errors
made in choice of disinfectants and cleaning procedures. Finally,
cleaning supplies make a difference when related to physical
removal of dirt, debris and contaminants. Guidance is provided in
Cleanroom Cleaning – Choosing, Using, and Handling Cleaning
Materials (Chapter 15) as to the choice of supplies as well as proper
use of these for adequate cleaning and disinfection.

It is our wish that this book is used as a guide to make

improvements, prevent, monitor and remediate contamination
issues using a holistic and science-based approach.

Ziva Abraham and Morgan Polen

Editors
 1

CONTAMINATION RELATED
CASE STUDIES AND
483 OBSERVATIONS

Morgan Polen and Ziva Abraham

Microrite, Inc.
San Jose, CA
USA

COST OF CONTAMINATION
Any contamination in the drug manufacturing process can have
a substantial financial impact, not to undermine possible safety
implications. Drug manufacturers making contaminated products
which may be harmful to patients are forced to halt production, recall
drugs and remediate issues, not to mention the public relations and
mistrust challenges. This may cost hundreds of millions in revenue
loss as well as diminished regulatory and public confidence. There
are further implications to producing contaminated products.
These may include years of litigation with the Food and Drug
Administration (FDA) ending in fines which may cost additional
millions. From the detected contamination event to commencement
of manufacturing the cost to an adulterated drug manufacturer may
be immense.

1
2 Cleanroom Contamination Prevention and Control

PRIMARY CAUSES OF CONTAMINATION EVENTS

As evident with the increasing number of warning letters, product
recalls and 483 observations related to both particulate and
microbial contamination, it is evident that contamination control
principles are either not well understood or ignored. The proposed
holistic contamination control strategy in the EU Annex 1 revision
is the first step; however, without thorough working knowledge of
each system a documented contamination control strategy becomes
another paper exercise.

A poorly designed cleanroom and/or a poorly integrated barrier

system coupled with inadequately executed smoke studies will
fail to identify the risk to product. This will lead to inappropriate
monitoring locations missing the real risk to product and patient.
Outdated cleanroom and barrier systems designs, and limited
understanding of the real airflows has led to repeat contamination
related observations worldwide. Not mitigating contamination
possibilities leads to cleanrooms and barrier systems which are
unsuitable for the manufacture of sterile products. Once a cleanroom
is built and the barrier system is installed, few manufacturers would
go back to modify the design. This is one of the most prominent
sources of contamination as well as data integrity. No amount of
Corrective and Preventive Actions (CAPAs) can mitigate such
situations; CAPAs are a futile exercise when these unfortunate
mistakes are made.

Patient risk is no longer understood as clinical microbiology has

become a discipline of the past. Additionally, most microbiologists
fail to keep up with emerging and re-emerging pathogens as well the
contaminant’s infectivity, especially for the immunocompromised
patient population which is growing exponentially.

The case studies and regulatory observations below will vouch

for the lack of knowledge and capability for keeping up with science,
technology and evolving organisms.
 Contamination Related Case Studies and 483 Observations 3

PARTICULATE CONTAMINATION
Particulate contamination control in microelectronics, device, as
well as medicinal product manufacturing environments is critical.
Control of submicron-sized particles is becoming increasingly
important, especially in microelectronics manufacturing operations
due to the continuing reduction in feature size. However, from
the numerous observations by regulators, the pharmaceutical
industry deals with macro particle contamination which should
be easier to control than sub-micron contamination; but is it?

One of the basic tenets of pharmaceutical quality is the

manufacture of drug products that are free of microbial, chemical,
and physical contaminants. Although microbial contamination of
injectable drug products is seemingly understood, defined, and
measurable, total particle control in drugs, medical devices and
drug device combination products is not well defined or recognized.
This is due, in part, to the nature of contaminants, the current state
of pharmaceutical manufacturing, and the availability of measuring
techniques, as well as the test methods which may not be sensitive to
all types and sizes of particles. These particles can be of various sizes
and configurations. Defining them as detectable by visual inspection
(in general ≥ 50 µm) or sub-visible inspection with a range of 2–50 µm
in size is the way particles are characterized in the pharmaceutical
industries. Hundred percent visual inspections are required per
the regulations, but there could be gaps in testing that may prevent
detection. USP <790> defines “essentially free” as criteria for a batch
of parenteral product that has been 100% inspected, meeting an
Acceptance Quality Limit (AQL) of 0.65% or tighter.

Universal tests and qualification of the inspection process should

be performed with reference to particulates in the visible range and
those particulates that might stem from the manufacturing or filling
process. Every container in which the contents show evidence of
visible particulates must be rejected.
4 Cleanroom Contamination Prevention and Control

Often visual inspections are performed by humans; people

are more limited in the rate of inspection due to the number of
containers per minute or hours that they can inspect. They also
suffer fatigue and require frequent breaks to maintain a high-
performance level. These limitations lead to greater variation in
manual inspection results.

For the determination of sub-visible particles, the Light

Obscuration Particle Count Test is the preferred method. Morph­
ology, number and refractive index of the particles, rheology and
opalescence of the solutions as well as other factors may hinder
detection of particles when inspections are performed manually but
also when using automation (Sharma et al., 2010). Flow microscopy
is an emerging technology which, when suitably optimized, can
provide a number of advantages over existing techniques in the
analysis of sub-visible particles.

The particles monitored during classification and environmental

monitoring (≥0.5 µm and ≥5.0 µm) and those checked during
product inspection for particulate contamination are starkly differ­
ent. Meeting non-viable particle levels, as set for environmental
monitoring per ISO (International Organization for Standardization)
standards, in the cleanroom or barrier does not guarantee particulate
free product. The equipment used for environmental monitoring is
not capable of detecting these size of sub-visible, particles (2 µm
to 50 µm) and visible particles (>50 µm). Particle contamination in
medicinal products can occur from extrinsic, intrinsic and inherent
particles.

Extrinsic particles are defined as those that are not part of the
formulation, packaging, or process, but are foreign and unexpected.
Examples of extrinsic particles include fibers (e.g., cellulous),
clothing fragments, hair, rubber, metal, plastic, paint, insects, etc.
Biological extrinsic particles such as skin flakes, insect parts and hair
are not tolerated by regulators.

Intrinsic particles are defined as those that arise from sources

related to the formulation, packaging, or processes. Examples of
 Contamination Related Case Studies and 483 Observations 5

intrinsic particle materials include glass, stainless steel, rubber from

stoppers, and gasket material; intrinsic particles require specific
considerations.

Inherent particles are defined as materials that are expected

from the drug formulation, and thus represent generally accepted
characteristic of the product. Examples of inherent matter such
proteinaceous aggregates are shown in Table 1.

Table 1 Particulate types and sources

Source Particulate Type

Environmental including personnel Dust Extrinsic
Fibers
Biologics such as insect parts,
pollen, organisms
Hair
Skin
Paint or coating
Rust
Metal
Minerals
Glass
Rubber silicone
Packaging material Rubber Intrinsic
Glass
Polymers
Silicone
Solution and formulation components Undissolved materials Intrinsic
Degradants
Agglomerates
Product-package interactions Glass lamellae Intrinsic
Silica
Rubber
Plastic

RISK TO PATIENT FROM PARTICULATES

The route of pharmaceutical product administration can influence
the deposition of the injected particles, the total particle load
administered to the patient, and the overall risk to the patient.
6 Cleanroom Contamination Prevention and Control

Many clinical effects have been documented in patients who have

received injections containing particulate matter contamination.
The size as well as the shape of the injected particle can affect
both its deposition within the body and its clinical effects on the
patient. Examples include phlebitis, pulmonary emboli, pulmonary
granulomas, immune system dysfunction, pulmonary dysfunction,
infarction, and death. For the intramuscular and subcutaneous
route of administration the risk of a systemic reaction is low, and the
ability of these particles to migrate far from the injection site may
be negligible. However, in case of vascular injections delivery of
greater volumes of fluids and greater dissemination and deposition
of particulate matter throughout the body is possible.

On the other hand, ocular administration, such as eye drops,

ointments, in-situ gels, inserts, multi-compartment drug delivery
devices, and ophthalmic drugs can cause great harm to the patient
(Langille, 2013).

The below noted recalls (Table 2) and regulatory observations

may highlight the challenges and shortcomings of the current testing
methods for particulates, but these should also be considered as an
impetus for proactive contamination control.

Table 2 List of recalls reported by FDA in 2014 (Tawde, 2014)

Product Manufacturer Reason

IV Sodium chloride and potassium Baxter Particulate matter (cellulosic
chloride solutions fibers and/or plastics)
CLINIMIX and CLINIMIX E Baxter Particulate matter
Injection
CUBICIN (daptomycin for injection) Cubist Particulate matter (glass
Pharmaceuticals particles)
Dianeal low calcium (2.5 mEq/L) Baxter Particulate matter (presence
Peritoneal dialysis solution with of oxidized stainless steel,
2.5 percent dextrose 5000 mL garment fiber, and PVC
(Ambu-Flex II) particulate matter)
Coumadin (warfarin sodium) for Bristol-Myers Particulate matter (metallic
injection Squibb and non-metallic cellulose
materia)
 Contamination Related Case Studies and 483 Observations 7

Product Manufacturer Reason

Dobutamine injection Hospira Visible particulates
(250 mg/20 mL)
Heparin sodium, 1,000 USP Hospira Particulate matter (human
heparin units/500 mL (2 USP hair)
heparin units/mL), in 0.9 percent
sodium chloride injection, 500 mL
Lidocaine HCI injection, USP 10 Hospira Particulate matter (human
mg per mL, 30 mL single-dose, hair)
preservative-free
Lidocaine HCI injection, USP, 2 Hospira Visible particulates (iron
percent oxide)
Marcaine (Bupivacaine HCI Hospira Visible particulates (glass
injection, USP), 0.5 percent, 30 mL, defect)
Single-dose, preservative-free vial
Marcaine (Bupivacaine HCI Hospira Visible particulates (glass
injection, USP) 0.25 percent, 10 mL, defect)
single-dose, preservative-free vial
Propofol injectable emulsion, USP Hospira Visible particulates (glass
defect)
0.9 percent sodium chloride Baxter Particulate matter
injection USP in 100 mL MINI-BAG
PLUS container
Soliris (eculizumab) concentrated Alexion Visible particulates
solution for IV infusion
VPRIV (velaglucerase alfa for Shire Particulate matter (stainless
injection) Pharmaceuticals steel and barium sulfate)
Labetalol hydrochloride injection Hospira Visible particulates (stainless
100 mg/20 mL (5 mg/mL), 20 mL, steel and iron oxide)
multidose vial

Many medical device manufacturers test for particulate matter on

their devices, and various extraction and measurement techniques
are used (Kielhorn, 2016). Some manufacturers may base their
methods on those designed for drugs, such as USP <788>, while
others modify the methods provided in one of the few regulatory
documents that address particulates in medical devices.
8 Cleanroom Contamination Prevention and Control

STATIC CONTAMINATION
Static electricity is caused by machinery where there is friction,
contact and separation, as well as in instances where there are rapid
heat changes. People can build up their own static charges simply
by the friction created during movement. Some other sources of
static electricity production can be liquid flowing through a pipe,
hose or opening, blending or mixing, spraying or coating, filling
drums, cans, pails, or tanks and conveyer belts among other sources.
All these are a part of various drug manufacturing processes.
Static control is not practiced in drug manufacturing but with ad­
vances in manufacturing technologies, use of robotics and drug
device combination, dissipating static charge should be a serious
consideration for contamination control.

MICROBIAL CONTAMINATION
Microbes live in almost every nook and cranny one can think of,
from miles beneath the earth’s surface to miles overhead. They
live at temperatures less than –20 degrees Celsius to temperatures
hotter than the boiling point. Microbes thrive on a huge range of
substrates including oil and toxic wastes. Every time one walks
on the ground billions upon billions of microbes lie underfoot.
Where there is water, there are microbes. Sources of microbial
contamination in pharmaceutical products include raw materials
used, environmental sources, cleaning equipment, packaging,
containers that are frequently re-used, repackaging of products,
processing, storage and transportation, among others.

Factors affecting spoilage of pharmaceutical products include

the size of contaminant inoculum, factors related to pH, moisture
content, storage temperature, packaging design, etc. Microbial
proliferation can be effectively prevented by proper storage and
use of preservatives. However, there are microorganisms that may
circumvent preservative effectiveness, and those products that do
not have preservatives or are organisms in nature are susceptible to
greater risk.
 Contamination Related Case Studies and 483 Observations 9

Whether dealing with particulates, microbial contamination

or static charge that will attract particles, proactive contamination
control begins with facility design, satisfactory airflows, meaningful
monitoring, optimal process controls and adequate testing.

SAFEGUARDING AGAINST CONTAMINATION

Because contamination may occur at any point in the manufacturing
and packaging process, no single step can be taken to eliminate the
risk of contamination in its entirety. This starts from facility design
and ends in final product testing and everything in between.

Everything from the facility’s layout and architecture, cleaning

supplies used, regulating air, personnel, material, and waste should
be determined with the goal of contamination prevention in mind.
Incoming materials, environment, utilities, process and product
should be monitored using methods that are effective at detecting
contamination. Airborne contaminants pose a serious threat as
well; so, the heating ventilation and air conditioning (HVAC)
system should be designed in such a way as to minimize the risk
of insufficiently treated air coming into contact with products on
the manufacturing line. Contamination control cannot be attained
by solely addressing the area where contamination has occurred –
a holistic approach is necessary. The purpose of this chapter is to
identify the causes of some repeat observations, recalls and warning
letters and derive the pattern of similar mistakes made by different
companies across the globe.

Guidance issued by regulatory agencies is loud and clear, but so is

the lack of science and foresight behind these guidances. This review
of the warning letters and case studies will offer a learning guide to
help avoid similar non-compliance challenges. Quality, compliance
and integrity are the pillars of any pharmaceutical organization
which wants to be successful across the board, including profitability
and patient safety. The examples and observations below prove that
if the basic tenets of contamination control are neglected, particle
and microbial contamination will ensue.
10 Cleanroom Contamination Prevention and Control

CASE STUDY 1
PARTICULATE CONTAMINATION AND
ELECTROSTATIC CHARGE IN MEDICAL
DEVICE MANUFACTURING
Problem
A medical device company which was manufacturing a tissue
derived critical vascular device struggled with macro particle (≥5.0
µm) contamination. The source of this contamination was from a
plastic (Teflon/PTFE) component used as part of the construction
of the device. The electrostatic properties of the (Teflon/PTFE)
component made particle removal difficult as the electrostatic field
created an attractive force drawing particles in from the surrounding
areas as well as a bonding force making particle removal difficult.
Electrostatic charge was suspected to be a concern, the consultants
originally hired to address the electrostatic charge problem also
sold ionizers. A considerable investment in overhead room-based
pulse DC ionization was made. Ionization was added to the entire
manufacturing ceiling (ISO Class 7 non-unidirectional air flow) as
well as the entire gowning/change room. This however did not reduce
particle contamination on the product. What the company failed to
understand is that it did not have adequate cleanroom and process
design for addressing electrostatic charge or effective measures
for controlling or monitoring particles. This gap in understanding
of electrostatics related to particle control and a suitable cleaning
process lead to loss of yield.

Because airborne particulate monitoring of the ISO Class 7 clean-

rooms was within the limits, it was presented with a false sense of
confidence, while the macro particle issue on the device persisted.
 Contamination Related Case Studies and 483 Observations 11

Discussion
Particles needed to be controlled at the source of generation; there
was only one operation that was generating the particles which
occurred when the particle generation material was manipulated by
operators during the manual manufacturing operation.

Controlling electrostatic charge in cleanrooms is a complex

process that must be addressed in facility design as well as the
manufacturing operation. An electrostatic envelope must be created
in the manufacturing area. The material choice for flooring, walls,
and worksurfaces must be addressed in the design phase, and
verified in the installation phase. The workstations needed to be
upgraded with a suitable (grounded) worksurface and operator
grounding via wrist-straps.

Lessons learned
The electrostatic properties of the product materials (Teflon/PTFE
(PolyTetraFluoroEthylene)) were such that additional localized
ionization for each operator/workstation was also required. The
original room-based pulse DC ionization was ineffective at removing
electrostatic charge and preventing electrostatic attraction which
caused particles to stick to the final product during manufacturing.
The use of fan-based ionizers in close proximity to the manufacturing
operations was required when handling the product materials
(Teflon/PTFE) to prevent this phenomenon.

Additionally, the electrostatic environment of the cleanroom

also needed to be qualified and maintained with daily, weekly and
monthly test intervals to ensure electrostatic charge is addressed
and does not impact product quality.
12 Cleanroom Contamination Prevention and Control

CASE STUDY 2
CLEANROOM DESIGN FOR MANUAL
ASEPTIC PROCESS – 483 OBSERVATION
Problem
The following observation is by the FDA to a manufacturer with
an intensively manual aseptically processed sterile product for human
use: “You have not established and followed appropriate written
procedures to ensure that your intensively manual aseptic process is
capable of reproducibly yielding sterile (b)(4) unit” (https://www.fda.
gov/inspections-compliance-enforcement-and-criminal-investigations/warn
ing-letters/sr-burzynski-manufacturing-facility-06132016). The above
regulatory observation emphasizes the importance of facility design
and airflows to product quality, a key to protecting the product from
contamination and saving the patient from harm. Figure 1 below,
is representative of such a scenario; where aseptic processing is
performed in an open cleanroom.

Figure 1 Open cleanroom

Source: Morgan Polen

 Contamination Related Case Studies and 483 Observations 13

Discussion
Open cleanrooms for aseptic or low bioburden operations are an
old concept better left in the 20th century. In comparison, even
restaurants with a salad bar or buffet have a barrier system in the
form of a “Sneeze Guard”.

With the industry inundated by warning letters, recalls, consent

decrees and harm to patients, such designs should not be used
as contamination risk is unpredictable and there could be many
contaminated products being manufactured in such facilities.
Sterility tests should not be the measure of safe products; a passing
sterility test of a small fraction of the batch does not in any way
indicate that the entire batch is contamination free. More often
than not, sterility test failures stop at laboratory investigations, or
manufacturing operation investigations. Rarely the facility design
and airflows are explored as the cause of microbial ingress during
filling. Cleanrooms and barrier systems are validated, and a passing
smoke study performed by a certifier or inexperienced personnel is
kept on file, providing a false sense of security during filling.

Lessons learned
Modern contamination control technologies such as barrier systems,
biological safety cabinets (BSCs), restricted access barrier systems
(RABS) take advantage of the contamination control effect of directed
HEPA filtered air flow, creating a sweeping action that protects
products from external contamination while removing contamin­
ation intrinsic to the process.
14 Cleanroom Contamination Prevention and Control

CASE STUDY 3
CLEANROOM AND EQUIPMENT DESIGN,
OPEN DOOR INTERVENTIONS
– 483 OBSERVATION
Problem
Warning letter with observations related to poor aseptic behavior,
facility and equipment design and inadequate media fills.

“Your aseptic filling equipment design, room space, protection of

the area and filling equipment where connections are made, and the
number of personnel present during filling operations are deficient.
Basic design deficiencies and manually intensive interventions in your
operation undermine the ability to maintain asepsis.” (https://www.
fda.gov/inspections-compliance-enforcement-and-criminal-investigations/
warning-letters/akorn-inc-568173-06132019)

Discussion
Aseptic manufacturing requires scientifically sound contamination
control technologies and practices. Contamination control technol­
ogy in the 21st century is a multi-disciplined field of applied science
that is in a state of continuous development. The “C” in cGMP
stands for “current,” requiring companies to use technologies and
systems that are up to date in order to comply with the regulations.
Systems and equipment that may have been “top-of-the-line” to
prevent contamination, mix-ups, and errors 10 or 20 years ago may
be less than adequate by today’s standards. The cleanroom and
integration of equipment and barrier systems must be addressed
in unison with personnel, material, waste and air flows. Everything
in the cleanroom and every movement has an impact on the overall
contamination control effect of the entire system. This is why Smoke
Studies (air flow visualization) and Aseptic Process Simulation
(media fills) are used for evaluation of the contamination control
effectiveness of the overall manufacturing system.
 Contamination Related Case Studies and 483 Observations 15

Lessons learned
All aseptic activity must be considered as part of the design phase
and evaluated during the qualification stage. It is not enough to have
good aseptic techniques if the efficiency of operator movements
related to equipment and ergonomics is not considered. Aseptic
operations must also be evaluated from a contamination control
perspective as well as the ease of each operation. The goal should
be in reducing unnecessary operator movements and if possible,
the complexity of these movements as well as the overall number
of interventions. Modern equipment design should allow for fewer
corrective interventions and more automated intrinsic interventions.

CASE STUDY 4
LIQUID FILLING LINE
– 483 OBSERVATION
Problem
A certain company received a warning letter for data integrity
related to microbiological data deviations. Environmental monitor­
ing (EM) data revealed consistent failures in the filling RABS, yet
the results from media fills, product sterility testing and personnel
indicated no growth. The consulting group hired to help with
warning letter remediation watched for data integrity during
environmental monitoring, which ended in multiple CAPAs and
extensive training to no avail. These CAPAs resulted in similar
remedial efforts with little difference in the number of excursions.
The consultants increased scrutiny on the manufacturing oper­
ators and the EM technicians by watching them perform their
functions. The company decided to lay off a majority of the aseptic
operators and EM technicians and recruited an entirely new team
of operators and technicians; the situation did not improve. This
move did not address the issues, just increased the cost for the
company by hiring more consultants. This scenario was persistent
for six months until the root cause described below was identified.
16 Cleanroom Contamination Prevention and Control

Discussion
The cleanroom was constructed with a Laminar Air Flow Unit
(LAF) suspended from the ceiling. The filling line was beneath
this LAF with an enclosure referred to as an open RABS. The LAF
extended over the open RABS as well as the area the operators were
working in. As the LAF was not coupled to the open RABS, there
was no differential pressure between the inside of the RABS and
the occupied area. Additionally, the room was too small to support
aseptic operations as the restricted room height placed the bottom
of the diffuser membrane just inches away from the top of the open
RABS frame and the top of a standing operator’s head.

Open passive RABS technology does not provide suitable

differential pressure and air flow condition between the critical
zone (Grade A) and the occupied zone (Grade B). Coupled with
the close proximity of operating personnel (see Figures 2 and 3).
Particulate and microbial contamination from operating personnel
should be expected inside the critical zone. Due to this poor design
the company chose to falsify media fill, sterility and environmental
monitoring data in order to ship products.

Figure 2 Open RABS under LAF

Source: Morgan Polen

 Contamination Related Case Studies and 483 Observations 17
Figure 3 Open RABS under LAF

Source: Morgan Polen

Lessons learned
Open passive RABS are an outdated technology and are only
marginally better at preventing contamination than open clean­
rooms. New or upgraded facilities should consider active RABS
(directly coupled to HEPA filters) closed RABS or isolators for
aseptic operations. In addition, the cleanroom/RABS design and
integration should be considered holistically to reduce the risk of
contamination to the product or patient. This integration should be
confirmed by performing Computational Fluid Dynamics (CFD),
before construction and installation as well as air pattern analysis
(smoke studies with conclusions) as part of the qualification.
18 Cleanroom Contamination Prevention and Control

CASE STUDY 5
TERMINALLY STERILIZED FILLING OPERATION
WITH MOLD CONTAMINATION
– 483 OBSERVATION
Problem
A company received a warning letter for having foreign matter
as well as mold in its terminally sterilized product. Filling was
conducted in a Grade A Open RABS that was under a ceiling
mounted LAF module in a Grade C background room.

Figure 4 Ceiling damage due to mold growth

Source: Morgan Polen

Water damage in the ceiling (upper right hand corner)

Excessive caulking around HEPA filter (indicative of mold contamination from
leaks in interstitial area)
 Contamination Related Case Studies and 483 Observations 19
Figure 5 Air from HEPA filter in Grade C being sucked
into the RABS inlet

Grade C Air HEPA Filter next to

Grade A LAF inlet. This creates a
“Short Circuit”. HEPA filtered air
intended to control contamination
in the occupied zone is robbed by
the air inlet for the LAF. The results
are poor clean air mixing in the
occupied Grade C Zone (Figure 5)

Source: Morgan Polen

Discussion
Upon researching, it was clear that previous regulatory visits had
observations related to water leaks in the ceiling in other locations of
the same building. Smoke study videos showed clear indications of
water damage in the ceiling as well as excessive caulking around the
Grade C HEPA filters (see Figure 4). In addition, the Grade C HEPA
filters were in close proximity to the air inlets of the LAF module,
creating an “air flow short circuit” (see Figure 5) where the clean
air supplied to the Grade C background is sucked into the Grade
A supply LAF. This robs the cleanroom of clean air mixing in the
occupied zone.

There is published evidence that some mold types are resistant

to disinfectants as well as hard to sterilize. Consistent exposure to
mold in such a scenario provides a guarantee of sterility failures
even if the product is terminally sterilized.
20 Cleanroom Contamination Prevention and Control

Lessons learned
In this case the integrity of the building had been compromised.
Once a water leak is discovered inside a medical product
manufacturing facility, it’s seriousness cannot be ignored. Short of
a fire or power loss, a compromised building integrity situation is
the most serious building emergency that faces a medical product
manufacturing facility. Water leaks equate to water damage and
fungal contamination. Water damaged ceilings and excessive
caulking around HEPA filters are examples of poor root cause
analysis related to mold/fungal contamination investigations.

The placement of HEPA filters, air inlets, exhaust systems,

cooling fans and air returns must be taken into consideration to
maximize the contamination control effect of HEPA filtered air. Just
as it is considered poor cleanroom design to have ceiling mounted
air returns, equally bad is the placement of LAF modules that steal
air from the surrounding occupied (Grade B/C) zones. This poor
integration of the barrier systems into the background cleanroom
significantly reduces the contamination control effectiveness of the
entire manufacturing line. By having uncontrolled mixing of air in
the occupied zone, and air supply points located above operators,
(LAF inlets) larger microbe carrying particles are suspended in
the occupied area for longer durations. In addition, these microbe
carrying particles have a higher probability of being deposited
on operators and equipment. Due to the chaotic nature of particle
generation and settling, intermittent EM excursions and sterility test
failures may plague a facility for years before an accurate failure
mode is discovered.
 Contamination Related Case Studies and 483 Observations 21

CASE STUDY 6
BARRIER SYSTEM DESIGN FLAWS
AND CLEANROOM INTEGRATION MISTAKES
Problem
A new isolator for aseptic liquid filling had several problems
identified in Factory Acceptance Testing (FAT) and Site Acceptance
Testing (SAT). Factory Acceptance Testing identified turbulent air
flow in the filling zone. Additionally, the preconfigured EM sampling
fixtures and probes for viable and non-viable monitoring locations
were positioned considerably far from the critical operations to hide
this design flaw. The equipment supplier’s justification for the EM
locations was explained. “That’s where we always put them, and we
have 100s of systems all over the world”.

Site Acceptance Testing and Air Flow Visualization (AFV)

studies demonstrated that during open isolator door interventions
for equipment set-up and assembly (i.e., stopper bowl installation)
outside air (Grade C) clearly enters the aseptic isolator and demon­
strated Grade C air washing over the operators into the isolator
and onto product contact surfaces. Even though the operators are
gowned in sterile garments during set-up and assembly operations,
for part of the set-up operation, the operators have to insert their
upper torso (head, shoulders and arms) into the isolator.

The supporting cleanroom for the isolator was not designed to

support aseptic operations. The background room was designated
as a Grade C environment; however, the room was not balanced; it
had too few air returns for the number of HEPA filters and a very
low air exchange rate.

Discussion
Barrier system and equipment suppliers may not have a
comprehensive understanding of contamination control, air flow
and environmental monitoring requirements. Unidirectional air
22 Cleanroom Contamination Prevention and Control

flow is a critical requirement for aseptic operations, regardless

whether open cleanrooms, RABS or isolators are used.

Additionally, environmental monitoring locations are supposed

to be risk-based. However, the equipment suppliers often pick
locations that may mask design flaws by placing the non-viable
isokinetic probes closer to the HEPA filter or on the opposite side of
the critical location in order to demonstrate low non-viable particle
counts during qualification and EM. Another tactic to mask poor
airflow in RABS and isolators is the use of water or nitrogen-based
equipment for performing FAT and SAT smoke studies. These
“cleanroom foggers” create a fog that is heavier than air and this
phenomenon can give the impression of unidirectional air flow
even if the isolator HEPA air supply is switched off. Thus, any dead
spaces, eddy currents or turbulence could go undetected.

Lessons learned
Having a well thought out and expert reviewed User Requirements
Specification (URS) with clearly stated requirements and testing
specifications is important. Additionally, having a comprehensive
Computational Fluid Dynamics (CFD) study of the barrier system
as well as the cleanroom and the integration of these two systems
helps avoid poorly designed barrier systems and cleanrooms meant
for critical operations.

CASE STUDY 7
AIRFLOW VELOCITY
– 483 OBSERVATION
Problem
“The airflow velocity inside critical areas of the aseptic processing
operations of Line (b)(4) was found unacceptable by FDA” (https://
www.fdalabelcompliance.com/letters/ucm243561). The documented
evidence of the in-situ air pattern analysis (smoke studies) reviewed
during the inspection confirmed this condition. Air velocities were
 Contamination Related Case Studies and 483 Observations 23

not uniform across the Grade A processing zone. Air velocities in

the same zone ranged from 40 FPM (feet per minute) to 150 FPM.
Air flow visualization studies demonstrated eddy currents at critical
locations and areas of excessive turbulence. Due to the design of
the facility, HEPA filter replacement was problematic and routine
filter integrity testing (leak testing) had created a situation where
repairing damaged filters was preferable to replacement; this over
time had degraded some of the HEPA filters.

Discussion
The contamination control effect of HEPA filtered air is paramount
for establishing a suitable environment for aseptic and sterile
operations. Facilities must be designed in such a way as to
accommodate suitable testing, maintenance and repair.

Lessons learned
Maintenance and testing of the cleanroom is critical to ensure
adequate quality of air required to process medicinal products.
Equipment maintenance challenges should be discussed at the
design phase and should be a part of the URS for cleanroom build.

CASE STUDY 8
IMPORTANCE OF ERGONOMICS
IN ASEPTIC PROCESSING
– 483 OBSERVATION
Problem
The below FDA observations for a sterile API manufacturer empha-
size the importance of ergonomics during aseptic manipulations. It
is unreasonable to give aseptic operators the responsibility of prac-
ticing flawless aseptic techniques while depriving them of the tools
to meet these expectations.
24 Cleanroom Contamination Prevention and Control
“Problem during the airflow analysis (smoke study) of aseptic
connections on your (b)(4) equipment inside the laminar air flow
(LAF) ISO-5 area, our investigator identified air flow disturbances
and turbulence. Under dynamic conditions, air did not sufficiently
sweep across and away from sterile connections, so the sterility of any
product processed under these conditions could be compromised.”

“Furthermore, in our review of the smoke study, we identified multiple

aseptic technique breaches during aseptic connection of the (b)(4)
equipment. Your equipment design and aseptic processing operator
competencies appear to contribute to the lack of unidirectionality.
Aseptic processing equipment should provide for appropriate
ergonomics that enable operators to reproducibly conduct aseptic
manipulations. In addition, it is critical that your aseptic processing
operators have the knowledge and skills to practice strict aseptic
techniques. Even operations that have been successfully qualified
can be compromised by poor operational, maintenance, or personnel
practices.” (https://www.fda.gov/inspections-compliance-enforcement-and-
criminal-in vestigations/warning-letters/wockhardt-ltd-495920-12232016)

Discussion
Per FDA’s 2004 Aseptic Guidance “The layout of equipment should
provide for ergonomics that optimize comfort and movement
of operators” (FDA, 2004). Per PIC/S Isolators used for Aseptic
Processing and Sterility Testing (PIC/S GMP Guide):

“Air change, laminar/turbulent, aseptic technique, and ergonomics …

The design of the isolator system should include consideration of air
change rate, the use of laminar, unidirectional or turbulent airflow,
the application of aseptic technique and risk of error due to human
fallibility. The rationale for the decisions taken should be documented.”

The consideration of ergonomics and its impact on aseptic

techniques is not well defined, characterized, understood and
documented during the design phase leading to many aseptic
technique related failures.
 Contamination Related Case Studies and 483 Observations 25

Lessons learned
The importance of ergonomics should be focused upon at the design
phase of the barrier, machine and process. Leaning over open com-
ponents and products during aseptic processing is not conducive
to contamination control. Additionally, the comfort of the operator
is paramount, and should be taken into consideration in the design
and URS phase. Operator fatigue develops if the operators are un-
comfortable while performing critical operations; this can result in
serious aseptic technique violations.

CASE STUDY 9
LACK OF SCIENTIFIC RATIONALE
IN ENVIRONMENTAL MONITORING
– 483 OBSERVATION
Problem
“Your firm failed to establish an adequate system for monitoring
environmental conditions in aseptic processing areas (21 CFR 211.42(c)(10)
(iv)”. (https://www.fda.gov/inspections-compliance-enforcement-and-criminal-
investigations/warning-letters/mylan-laboratories-limited-464863-08062015)

“a. You do not have a scientific rationale for the environmental

monitoring sampling locations in aseptic filling suites (b)(4). You did
not include factors such as smoke study findings, number and location
of operators, and historical microbial data in your assessment of
hazardous points.”

“For example, we found that settling plates are not appropriately

placed in critical areas. Your smoke study showed that during set-
up and filling, air flows toward the front (when the (b)(4) is open)
or back of the RABS. However, two relevant sampling points were
recently eliminated. As a result, these points of increased risk are not
monitored.” (Office of the Commissioner)
26 Cleanroom Contamination Prevention and Control

Discussion
Environmental monitoring is not an exercise; it is an important tool
to predict risk to product and patient. To capture environmental
contaminants such as particulates and microbes, there are a myriad
of aspects of the environmental monitoring program that should be
considered. It starts with understanding the regulations, standards
and guidance that relate to environmental monitoring and not focus
on publications and hearsay. It is not uncommon that monitoring
locations are pre-determined in RABS and isolators prior to assess­
ing operational risks. Companies depend upon suppliers to select
sampling sites in these critical environments where for a supplier it is
just a feature in the barrier system. These hard-wired sampling sites
are not based on smoke studies, where there could be deviant air,
dead air or eddy currents. Additionally, the sites may not be proximal
to critical open operations and open product, hence do not allow
con­tinuous risk assessment during aseptic operations. Selection of
sam­pling sites directly under HEPA filters may assist environmental
monitoring data to look good but will not be conducive to detecting
contamination and risk to product and patient. Sampling sites should
be based on scientific judgment utilizing smoke studies, taking into
consideration operator interventions and proximity to open product.

Lessons learned
A science based environmental monitoring program is multi-faceted.
Properly performed smoke studies validate the contamination control
effect of the cleanroom/barrier system design and airflows. Where
airflows are not optimal, sampling provides a glimpse of possible
risk to product. Using the relevant standards and guidance ensures
meeting cleanroom classification and setting correct limits. Choice
of mon­itoring equipment, maintenance and calibration of this equip­
ment provides confidence in the results. Incoming quality testing
of media ensures that contamination, if present, will be recovered.
Lastly, trending data deviations and especially micro­organisms
provide valuable information on changes to be made in cleaning,
disinfection and flows which helps assess direct risk to patient.
 Contamination Related Case Studies and 483 Observations 27

CASE STUDY 10
NON-INTEGRAL CLEANROOM MATERIALS
– 483 OBSERVATION
Problem
“Aseptic garments worn in the filling area were also non-integral. We
observed 7 of (b)(4) sterile gowns with tears or holes; 8 of (b)(4) had
loose threads. We observed 2 of (b)(4) sterile hoods with tears or holes;
12 of (b)(4) had loose threads. We observed 8 of (b)(4) sterile booties
with tears or holes; 11 of (b)(4) had loose threads.” (https://www.fda.gov/
inspections-compliance-enforcement-and-criminal-investigations/warning-
letters/mylan-laboratories-limited-464863-08062015)

Discussion
Personnel and their activities are one of the major sources of
contamination in a cleanroom. Therefore, making an educated choice
for an appropriate gowning system is crucial in limiting human
borne contamination that can affect products or processes in the
cleanroom. The end user should understand required performance
criteria for gowns, test methods, and procedures for gowning
systems. These should detail garment choice criteria, garment
use, garment maintenance, as well as develop a quality control
program for managing cleanroom garments. It is prudent for the
end user to understand garment-related factors that may influence
the performance of cleanroom operations. Selection, construction,
material characteristics, performance, laundering, maintenance,
validation, and documentation, as well as applicable test methods
should be understood by the end user in order to evaluate relevant
properties of gowns for their cleanroom applications. According
to Ljungqvist and Reinmüller (1993) a fully gowned operator in an
aseptic area still sheds 10,000 particles per hour. The key reasons
for increase in human borne contamination is wearing large gowns
and over laundering gowns till they fray. Use of wrong detergents,
high temperature during laundering and drying or ironing gowns
compromise gown quality, increase pore size, generate particles and
render gowns a poor barrier.
28 Cleanroom Contamination Prevention and Control

Lessons learned
If personnel gowns are not adequately chosen and managed or
personnel gowning practices are sub-par, human borne contam­
ination will prevail.

Gown choice is often made by the purchasing department and

depends upon cost benefits; cost benefits of reducing investigations
and protecting product are ignored. The details about gown
materials, construction, laundering and maintenance is not well
understood nor con­sidered seriously. Additionally, this information
is only found in IEST RP CC003.4. IEST is not an organization
that pharmaceutical professionals attend or participate in, hence
this valuable information is not known or disseminated by many
industry leaders.

CASE STUDY 11
MISUSE OF DISINFECTANTS
Problem
Company found Chaetomium globosum an ascomycetous fungus
on the belt of the filling line which was proximal to open product
during aseptic filling operations. The organism was tested against
a peracetic acid (PAA) and hydrogen peroxide chemistry which is
sporicidal in nature to determine the efficacy against this mold. At
the manufacturer recommended dilution the PAA chemistry failed
to kill the fungus. A decision was made to double the concentration
of the agent and disinfect the entire facility. Using this high
concentration became the new disinfection procedure for the facility
leading to flaking of paint and compromised surfaces (Figure 6).

Discussion
The dilution factor for the label of a disinfectant is established
after testing for efficacy against microorganisms but also for
material compatibility and many other factors. In this case the
 Contamination Related Case Studies and 483 Observations 29
Figure 6 Compromised walls

Source: Picture (RF) purchased from Dreamstime

PAA chemistry was not compatible with multiple hard surfaces

at the higher concentration. This compatibility data is available
with the disinfectant manufacturers and suppliers as well as on
Environmental Protection Agency (EPA) website.

Lessons learned
Disinfectants are to be used per the manufacturer’s recommendations
as extensive testing is performed when a disinfectant supplier
registers the disinfectant with the EPA. Information on efficacy,
material compatibility, toxicity and more is readily available.
Disinfection and cleaning go beyond filling buckets and mopping. It
is a science and should be treated as such.
30 Cleanroom Contamination Prevention and Control

CASE STUDY 12
DISINFECTANTS MISUSE
Problem
A tissue-based company which performed all its aseptic operations
in BSCs struggled with contaminated tissue batches. The cleaning
of BSCs was performed only with a phenolic compound; phenolic
and quaternary ammonium compounds are known for leaving
residue.

Discussion
Phenolic and quaternary ammonium compounds are known for
leaving residue. The front grill in a BSC is an inlet for room air,
keeping the interior of the BSC where aseptic operations occur safe
from contamination (Figure 7). If this grill is blocked with residue,
the air bounces off the grill and lands right on the aseptic product
being manipulated within the BSC (Figure 8).

Figure 7 Airflow in a biological safety cabinet

Source: Courtesy of Acumen Technologies

 Contamination Related Case Studies and 483 Observations 31
Figure 8 Blocked BSC grill due to phenolic residue

Source: Ziva Abraham

Lessons learned
A cleaning and disinfection program should be developed with
an in depth understanding of the function and effectiveness of the
disinfectants and residue removal should be a part of a company’s
cleaning and disinfection program.

CASE STUDY 13
NONTUBERCULOUS MYCOBACTERIA IN
PHARMACEUTICAL WATER SYSTEM
Problem
A company recovered Mycobacterium chelonae group of non-
tuberculous mycobacteria in media fill samples. These organisms
were not observed in water testing or environmental monitoring.
32 Cleanroom Contamination Prevention and Control

Discussion
Adhesion and biofilm formation abilities of various Gram-negative
bacteria is observed in pharmaceutical water systems; the presence
of non-tuberculous mycobacteria is not commonly discussed. The
presence of non-tuberculous mycobacteria is known in hospital
water systems and dreaded in medical devices because of its
capability to circumvent certain disinfectants.

Non-tuberculous mycobacteria (NTM) have been found to

be ubiquitous in the environment and have been isolated from
numerous water sources, including wastewater, surface water,
recreational water, ground water and tap water. Piped water
supplies are readily colonized by mycobacteria thus biofilms may
serve as a reservoir for these opportunistic pathogens. NTM are
common environmental organisms and are recognized as being
difficult to remove from water systems (Baird et al., 2011).

Since the water system in this case stood idle for an extended
period, biofilm formation was inevitable. Additionally, as these
organisms require longer incubation periods, they were not detected
until they had the opportunity to present in media fill samples
where incubation time is longer than that for routine environmental
monitoring and water sampling.

Lessons learned
If water systems are not designed, maintained and tested using
adequate methods for detecting contamination, biofilm formation
is certain and organisms that pass through 0.2 micron filters may be
present in the process water and yet not detected due to inadequate
growth media or incubation (Martin). Most water borne organisms
are pleomorphic in nature and a few such mycobacteria and some
other Gram-negative bacteria are known to pass through filters due
to their minuscule size.
 Contamination Related Case Studies and 483 Observations 33

CASE STUDY 14
MONITORING EQUIPMENT
Problem
An MHRA inspector cited a company on particle monitoring;
asking the company to prove that they were not losing particles in
the long and coiled tubing of the particle monitoring device in the
Grade A RABs.

Discussion
Testing performed showed that over 40% of ≥5.0 micron particles
were lost validating the inspector’s observation. ≥5.0 micron particles
do not carry well due to their size, hence per ISO 14644-1:2015,
sampling tubing for ≥5.0 μm particles should be 1 meter or less and
the bend radius of any bend should not be over 15 cm.

Lessons learned
Particle monitoring is critical for cleanrooms, loss of particles during
collection may skew the results of environmental monitoring data,
thereby leading to incorrect conclusions about the cleanroom
environment.

CASE STUDY 15
HMI EXHAUST FAN AS SOURCE
OF BIOFILM FORMATION IN
MANUFACTURING EQUIPMENT
Problem
Manufacturing equipment contamination issues went unresolved
after multiple cleaning agents and cycles as well as repeated cleaning
validation studies.
34 Cleanroom Contamination Prevention and Control

Discussion
While conducting smoke study in the room it was discovered that
the air from the Human Machine Interface (HMI) exhaust fan was
blowing on the aseptic connection of the equipment. After directing
the exhaust to the room return duct and a successful cleaning cycle,
the contamination issues did not reappear.

Lessons learned
When investigating any microbial contamination, whether
within a cleanroom environment or equipment, the source of
contamination should be determined utilizing root cause analysis.
An investigative smoke study utilizing appropriate equipment
and media has proven to be an extremely useful tool in identifying
contamination pathways.

CASE STUDY 16
MANUFACTURING EQUIPMENT
CLEANING VALIDATION AND
PRODUCT TESTING FAILURES
Problem
A manufacturer for sterile fluids had complaints from the clinics that
the fluid was hazy when received. The fluid was a sodium chloride
solution. There were many complaints related to this as well as
recalls and regulatory actions.

Discussion
An audit of the manufacturing facility and the microbiology labor-
atory revealed various shortcomings in contamination control
and prevention.
 Contamination Related Case Studies and 483 Observations 35

The isolates recovered from the product were identified using

three microbial identification systems; a mixture of genotypic and
phenotypic identification technologies. A total of 18 unique Gram-
negative genera and species were identified. Considering that the
solution was a salt solution, only two of the halophiles identified
should have been able to grow in this chemistry. High salinity of
the product represented an extreme environment to which relatively
few organisms have been able to adapt and occupy. After sending
the product to a qualified laboratory the results confirmed that only
two halophile species were the contaminants.

Media used for sterility testing of this salt solution were not
capable of growing these halophiles, hence the sterility test passed
each time, but the organisms continued to proliferate in the product.
Process investigation showed that filling needles were plugged with
biofilms. Transfer tubing from the final bulk to the filling apparatus
was slimy on the interior and the mixing tanks were riddled with
biofilm formation. Cleaning validation had failed. All the final rinse
bioburden testing for cleaning verification passed as the media used
were not capable of growing the contaminating halophiles.

Lessons learned
Though this a severe case of cleaning validation shortcomings,
The chemical verification of cleaning validation is relatively well
described, though it is important to note that more often than not,
the microbial aspect of cleaning validation is ignored... Important
to the microbiological aspect of cleaning validation are micro­
organisms themselves (a direct hazard) and the presence of
residues that potentially provide a microbial growth source, should
contamination be present, or contamination occur during the hold
period (an indirect hazard).
36 Cleanroom Contamination Prevention and Control

CASE STUDY 17
CONTAMINATION IN AN ISO
CLASS 8 PURIFICATION ROOM
Problem
Mold was consistently recovered in ISO 8 Purification Room during
environmental monitoring.

Discussion
When the mold contamination was discovered in the ISO 8
purification room during environmental monitoring, the company
decided to change HEPA filters in the room, and walls were broken
down and reconstructed in hope of finding the source. Upon review
of environmental monitoring data, it was observed that the same
mold was recovered in the ISO 8 corridor leading to the purification
room, the mold was also recovered in the materials airlock but not in
the gowning area. This investigation led to the carboys of materials
being stored in coldrooms; monitoring of the coldrooms recovered
the same mold. The company lacked wipedown procedures while
bringing in carboys through material airlocks into the cleanrooms.
There were two such purification rooms with similar processes, but
the second purification room did not present a similar issue as each
carboy/container was wiped down with a peracetic acid chemistry
followed by 70% sterile Isopropyl Alcohol (IPA).

Lessons learned
The solution to this contamination issue would have been simple if a
science-based approach would have been used and the mold would
have been tracked back to its origin instead of making assumptions.
 Contamination Related Case Studies and 483 Observations 37

CASE STUDY 18
PRODUCT CONTAMINATION
Problem
An in-vitro diagnostic company had its reagent cuvettes growing
mold. The company was a contract manufacturer, and many lots of
the diagnostic reagents were affected. Per the sponsor’s instructions,
the CMO fogged the facility, and increased environmental
monitoring; this presented no difference in the mold types and
quantities recovered inside the reagent. The sponsor started a
germination study for the two of the mold species recovered, the
study showed that both the mold species, Cladosporium cladoporoides
and Aureobasium species were capable of growing in the product
(reagent).

Discussion
The design of the facility was such that the bulk manufacturing area
was positively pressurized to the filling area. The filling area was a
makeshift ISO class 5 curtained barriers with HEPA filters. The far
end of the filling room was used as a packaging area, with pallets,
cardboard, paper and forklifts moving between the packaging area
and the warehouse. The door to the filling/packaging room was
perpetually open and was right across the warehouse. Additionally,
there were no wipe down procedures for the bulk carboys which
were stored in 10 coldrooms in the facility. The coldrooms had not
been cleaned since the facility was built, approximately 12 years
before. All the mold species found in the product were present in the
coldrooms as well as on carts used for transporting bulk containers.
Labels of raw materials and reserve samples in the coldrooms had
black borders due to mold growth on the glue. Mold growth hung
from the ducts in the coldrooms and resembled spider webs; even
the chromatography columns had the same mold growth in them.
38 Cleanroom Contamination Prevention and Control

Lessons learned
Facility design and maintenance is important even in non-sterile
or low bioburden product manufacturing. This is also an excellent
example of organisms circumventing antimicrobial activity, and
sheds light on the fact that the mold tested for USP antimicrobial/
preservative efficacy test may not be the best choice to determine
antimicrobial activity in the product. There are organisms that are
more resilient than those used for USP antimicrobial/preservative
efficacy testing. If any mold beyond those tested for USP anti­
microbial/preservative efficacy are recovered in the product, even in
small quantities, they must undergo this testing. These organisms
may have adapted or may be utilizing the product as a food source.

CASE STUDY 19
BOOKLICE IN STABILITY CHAMBER
Problem
“Booklice” is not a word in a pharmaceutical professional’s
vocabulary. They are wingless insects; and because they often live
among books and papers, they are usually called “booklice”. They
may make their way into laboratories and in this case a stability
chamber. These critters were happily strolling through shelves, on
containers, but mostly congregated near the labels.

Discussion
These booklice were seen for the first time in the stability chamber
in question. On inspection it was obvious that one side of the
chamber had containers with plastic labels which were snug,
while the stability samples on the other side of the chamber bore
paper labels. Upon close examination, it was noticed that the paper
labels were peeling off and there were pockets formed where the
booklice resided. Booklice live in areas where there is likely to be
high moisture content in the air; so, there was no better place than
a high humidity stability chamber for these critters. Booklice are
 Contamination Related Case Studies and 483 Observations 39

known to consume mold; as mold proliferates on glue and paper,

this combination of humidity and a food source generated a perfect
environment for a booklice habitat.

Lessons learned
Many coldrooms and stability chambers make for ideal environ­
ments in order for mold to proliferate. Introducing additional
sources which may constitute food for mold should be avoided in
these rooms.

CASE STUDY 20
CONTAMINATION OF INCUBATORS
Problem
A cell therapy company had testing failures with Gram-negative
bacteria; since the batches made were patient specific, this was a dire
issue and a quick resolution to this contamination was required.

Discussion
Incubators used for growing human and animal cells are CO2
incubators with direct heat (air jacket) or water jacket configuration.
They are considered an optimal solution for tissue cell culture
growth by properly controlling temperature, humidity, CO­2 gas,
and sterility. For constant humidity control a unit with a container
filled with water and electronic controls are used. In this case the
company followed the manufacturers instruction for cleaning the
water containers and connective tubing. However, they did not
follow the frequency of cleaning suggested by the manufacturer. Per
the manufacturer’s recommendation they used quaternary ammon­
ium compounds which was not sterile. Quaternary ammonium
compounds are known for supporting growth of Gram-negative
bacteria; there have been recalls for QACs due to growth of Gram-
negative bacteria (Adair et al., 1969; Sundheim et al., 1998).
40 Cleanroom Contamination Prevention and Control

Lessons learned
Incubators are excellent breeding grounds for mold and Gram-
negative bacteria as they provide an ideal growth environment
due to humidity and optimal temperature. Multiple transfers of
cell cultures from BSCs to incubators provides ample opportunity
for contaminating the cells by operators whose gloved hands get
in contact with the outside of the cell culture flasks which may be
contaminated. Maintaining cleanliness in the incubators is critical,
especially for cell therapy products as each batch consists of multiple
transfers and incubations.

CASE STUDY 21
LABORATORY RELATED CONTAMINATION
Problem
Bioburden testing of a non-sterile product consistently exceeded
specification.

Discussion
The laboratory was equipped with two horizontal laminar flow
hoods one across the other. After multiple data deviations related
to product testing from one LAF, the laboratory manager decided to
switch hoods to no avail.

Figure 9 below depicts that with the air flowing horizontally

two things are happening.

• The air bounces against the analyst and is directed back; most
probably carrying the contamination from the analyst onto the
product. Additionally, laboratory coats are often not changed
frequently in non-sterile testing.

• Horizontal air from one hood may carry contaminants from the
operators into the opposing hood.
 Contamination Related Case Studies and 483 Observations 41
Figure 9 Behavior of air in horizontal flow hood

Source: Morgan Polen

Lessons learned
Horizontal flow hoods are not adequate for many applications,
instead BSCs should be used for microbiological testing.

CASE STUDY 22
MICROBIOLOGY MEDIA
Problem
A sterile manufacturing facility had mold growth in a settle plate
sample from the Grade A RABS. This was not the first time they
had seen mold in Grade A, B and other controlled environments.
Consultants were engaged, leading to performing disinfectant
qualification studies, repeating smoke studies, revamping the
environmental monitoring program and retraining personnel.

Discussion
Close examination of the media packages and individual plates
threw all the prior root causes out the door. It was observed that
many plates had media at the edge between the plate and the lid.
42 Cleanroom Contamination Prevention and Control

This showed that the incoming quality control of media was not
robust. The checks were for dry media, compromised plates, color
difference, pits or bubbles, but not media in and on the lids. Most
conventional microbiologists are well aware of this phenomenon
and how it occurs. When media plates with seemingly solidified
agar gel are inverted, such leaks occur; the agar may be still liquid
underneath. This leaked agar streams down on the inner side of the
plate onto the lid; and solidifies. Such compromised media plates
may capture contaminants from the environment as well as during
incubation as media plates are inverted.

Lessons learned
Culture media are of fundamental importance for all microbiological
tests. Managing of culture media quality precedes growth promotion
testing. Multiple 483 observations have been issued related to dried
or cracked media; however not much attention is paid to the false
positive results due to improper media preparation and pouring.
Figure 10 is a good example of how environmental mold can start
growing between the plate and the lid if the media is not poured or
dried correctly, giving rise to false positive results.

Figure 10 Mold growth starting on the edge of lid and

subsequently in the plate

Source: Ziva Abraham

 Contamination Related Case Studies and 483 Observations 43

CONCLUSION
Today’s regulatory environment greatly encourages taking
a holistic approach to contamination control. This requires a
mentality that incorporates a holistic approach to contamination
control for it to be successful. Bringing together the perspectives
from various aspects of GMPs: from facility design to products
release can broaden our understanding of how to prevent con­
tamination and connect the dots between different systems and
the effect of each system as well as overall quality. This holistic
contamination control mindset plays a key role in developing
and producing a safe product with reduced contamination risk.
All the above case studies provide valuable information on how
deficiencies in one system can affect other systems; for example,
bad cleanroom or barrier system design may lead to environmental
monitoring excursions or media fill failures. Mismanaged gowns
may lead to environmental monitoring or product failures, and
lack of control on microbiological media may lead to false negative
or false positive data.

REFERENCES
Adair, F.W., Geftic, S.G., Gelzer, J. (1969) Resistance of Pseudomonas
to Quaternary Ammonium Compounds. I. Growth in
Benzalkonium Chloride Solution. Journal of Applied Microbiology
18(3): 299–302.

Baird, S.F.,Taori, S.K., Dave, J., Willocks, L.J.. Roddie, H., Hanson, M.
(2011) Cluster of non-tuberculous mycobacteraemia associated
with water supply in a haemato-oncology unit. The Journal of
Hospital Infection US National Library of Medicine. Accessed
July 15, 2020. https://pubmed.ncbi.nlm.nih.gov/21899922/

EU GMP (2011) Annex 1: Manufacture of Sterile Medicinal Products.

Revision November 2008.
44 Cleanroom Contamination Prevention and Control

FDA (2004) Guidance for Industry Sterile Drug Products Produced

by Aseptic Processing – Current Good Manufacturing Practice.

IEST-RP-CC003.4. ANSI Webstore. Accessed July 15, 2020. https://

webstore.ansi.org/standards/iest/iestrpcc003-1441187?gclid=CjwKC
Ajwr7X4BRA4EiwAUXjbtzzrYur8yL8L6GaYT4lc5zFfjGpoM9mC6
pWd7KJ351Y9buDfSmNdfxoC-2AQAvD_BwE

ISO 14644-1:2015 Cleanrooms and associated controlled environ­

ments. Part 1: Classification of air cleanliness by particle
concentration.

Kielhorn, C.M. (2016) Regulating particles on implantable medical

devices.” IVT Network. https://www.ivtnetwork.com/article/regulat
ing-particles-implantable-medical-devices

Langille, S.E. (2013) Particulate matter in injectable drug products.

PDA Journal of Pharmaceutical Science and Technology 67(3): 186–
200.

Ljungqvist, B., Reinmüller, B. (1993) Interaction between air

movements and the dispersion of contaminants: clean zones
with unidirectional air flow. Journal of Parenteral Science and
Technology 47(2): 60–69.

Martin, A. “Identification of Mycobacteria.” Millipore Sigma.

Accessed July 15, 2020. https://www.sigmaaldrich.com/technical-
documents/articles/microbiology-focus/mycobacteria-identification.
html

Office of the Commissioner “Warning Letters.” US Food and Drug

Administration. Accessed July 15, 2020. https://www.fda.gov/
inspections-compliance-enforcement-and-criminal-investigations/
compliance-actions-and-activities/warning-letters

PIC/S GMP Guide (PE 009-11) Annex 1 ClauseL 3, 53, 54.

 Contamination Related Case Studies and 483 Observations 45

Sharma, D.K., King, D., Oma, P., Merchant, C. (2010) Micro-flow

imaging: Flow microscopy applied to sub-visible particulate
analysis in protein formulations. The AAPS Journal. https://www.
ncbi.nlm.nih.gov/pmc/articles/PMC2895433/

Sundheim, G., Langsrud, S., Heir, E., Holck, A.L., Bessems, E.,
Terpstra, P.M.J. (eds.) (1998) Bacterial resistance to disinfectants
containing quaternary ammonium compounds. International
Biodeterioration & Biodegradation 41(3–4): 235–239.

Tawde, S.A. (2014) Particulate matter in injectables: Main cause for

recalls. Journal of Pharmacovigilance 3: e128.

ABOUT THE AUTHORS

Morgan Polen is a subject matter expert in the field of contamination
control, airflow visualization and particle monitoring in cleanrooms.
Morgan has over 35 years of industry experience working in
cleanrooms in all industries.

Morgan’s Technical Leadership:

• Member of International Disk Drive and Equipment Materials
Association (IDEMA) Standards Committee.
• Member of US Technical Advisory Group to ISO Technical
Committee 209 (international cleanroom standards i.e., ISO
14644 Cleanrooms and Associated Controlled Environments).
• Member of ASTM Subcommittee E55.06 Working Group “Guide
for Critical Airflow Visualization”.
• Board member of the Institute of Environmental Sciences and
Technology (IEST).
46 Cleanroom Contamination Prevention and Control

Morgan has been instrumental in drafting and editing international

cleanroom standards, contamination control guidelines and best
practices. He has extensive experience working on cleanroom
projects in the United States, Canada, Mexico, Germany, Malaysia,
Taiwan, South Korea, Singapore, Thailand, United Kingdom, Ireland,
China, Philippines, India and Turkey and is a valuable resource in
addressing contamination control in critical environments for the
electronics, aerospace and healthcare industries.

As a key member of Microrite’s Expert Contamination

Control team, Morgan is instrumental in development of proactive
contamination control strategies through pragmatic risk assessment,
particle testing and airflow visualization. Morgan’s broad experience
troubleshooting contamination issues has helped companies with
FDA 483/warning letter remediation.

Ziva Abraham has over 35 years of academic, research, clinical and

industrial experience in Microbiology, and Quality Assurance. Ziva
has received her master’s degree in Microbiology and has conducted
research on developing Microbial Insecticides using entomogenous
bacteria and fungi towards her Ph.D. degree.

She worked as a clinical scientist for many years and established

clinical laboratory systems in Israel. In this capacity Ziva evaluated
the first automated microbial identification system, introduced
new technologies for HIV testing, and many rapid testing methods
into the group of laboratories she helped establish and manage
for Maccabi Medical. These laboratories conducted clinical
testing in microbiology, chemistry, parasitology, hematology and
immunology.

She is a passionate microbiologist and mycologist and provides

training worldwide on various microbiology and contamination
control topics. Her clinical experience helps her evaluate and
teach the clinical implications of microbial contamination on
patients. Ziva is passionate about fungi; as in her research she had
 Contamination Related Case Studies and 483 Observations 47

collected, identified, and tested hundreds of species. She routinely

teaches hands-on fungal identification as well as investigation
and remediation of mold contamination and most importantly the
emergence of new fungal infections.

Ziva established Microrite, a California based consulting firm,

in 1998 as an independent microbiology consultant. Ziva used her
extensive research experience with fungi and clinical organisms
and their impact on human health to build an internationally
renowned contamination control team of experts. Her team consists
of facilities, cleanroom, airflow, static charge, validation, quality and
microbiology professionals with practical knowledge in their field
and involvement in industry standards and guidance organizations.
Ziva’s team works congruently to resolve client issues, prevent
contamination through design and help clients produce safe
products for human and veterinary use.
 2

PAPER COMPLIANCE VS FACTUAL

CONTAMINATION CONTROL

Ziva Abraham
Microrite
San Jose, CA
USA

A risk management program starts with identifying the possible

risks associated with a product or with the process used to develop,
manufacture, and distribute the medicinal product. Patient risk
from contaminated products is the main premise for risk assess­
ments and quality risk management. The only alternative to risk
management is crisis management and crisis management is more
expensive, time-consuming and embarrassing. Risk management
is not about creating long, complex measures and piles of paper; it
is about identifying what can go wrong and establishing practical
steps to protect the patient. Ignorance and indifference are ones
worst enemies, and the attitude of complacency, despite knowing
something is not right, leads to risk mismanagement. Most disasters
occur when there was an obvious warning of what could occur,
but management failed to act (Frank et al., 2008). Most root causes
are related to weaknesses or breakdowns in management systems.
Mishaps occur due to a lack of knowledge, failure to use the
knowledge or inability to seek knowledge. Many businesses think

49
50 Cleanroom Contamination Prevention and Control

that undertaking risk assessment is a difficult and complicated

process, and as a result, it is often avoided. This is the reason why
many companies employ third party consultants, at times working
from templates, without real knowledge of all aspects of client
process or product, to perform a series of assessments and prepare the
required documentation. This documentation is neither understood
nor read by anybody in the company and is finally deposited in a
remote cupboard/bookshelf never to be opened/read until the next
audit. Risk assessments must be more than a paper exercise and must
be taken seriously as the end user is a fellow human being. Hazard
identification and risk assessment methodologies vary greatly across
industries, ranging from simple assessment to complex quantitative
analyses with extensive documentation. There is no known magical
tool that will identify all risks.

The quality of risk assessment depends upon risk appetite

and risk tolerance of management first, followed by availability of
credible resources, information, training, experience, support, time
investment, involvement, motivation and interest of the team, as
well as the team’s composition.

The FDA (Food and Drug Administration) has identified a

risk-based orientation as one of the driving principles of the cGMP
(Current Good Manufacturing Practice) initiative. This reflects the
FDA’s commitment to the adoption of risk management principles
which enhance the Agency’s inspection and enforcement program,
which is focused on protecting the public health (FDA, 2004).

The concept of pharmaceutical QbD (Quality by Design) is a

systematic approach to development that begins with predefined
objectives and emphasizes product, process understanding, and
control, based on sound science and quality risk management (Yu et
al., 2014). The goals of pharmaceutical QbD include:

• Achieving meaningful product quality specifications that are

based on clinical performance.
 Paper Compliance vs Factual Contamination Control 51

• Increasing process capability and reducing product variability

and defects by enhancing product and process design, under-
standing and control.

• Increasing product development and manufacturing efficiencies.

CONTAMINATION RISK
The term contaminants include any unwanted matter that is found
in the product. These contaminants may affect the quality of the
product or the process or may cause harm to patient.

There are four general contamination types:

• Physical contamination which may include fibers, insects, metal,

and any type of particulate matter.

• Chemical contamination such as vapor, gasses, moisture, and

molecules.

• Biological contamination such as fungus, bacteria, virus as well

as actinomycetes (which are not routinely tested).

• Cross contamination which is unwanted matter which could

be residual product or biological contamination introduced or
brought from one process to the next during manufacturing.

The most underestimated risk is that of emerging and re-emerging

infections when it comes to biological contamination. The more we
learn about microbial genetics, structure, and function, the more we
marvel at the sophistication of the survival strategies of said microbes.
Their mechanisms of survival are many and varied, and specific
pathogens are generally tailored to flourish in particular niches.
Not only do microbes evolve much more quickly than humans,
but their enormous evolutionary potential is further enhanced by
their sheer numbers as well as their many ingenious mechanisms
of gene exchange (e.g., conjugation and plasmid interchange, etc.).
There is a vast and largely un-characterized pool of possible human
52 Cleanroom Contamination Prevention and Control

pathogens and the increasing opportunities for infection presented

by ecological upheaval, increasing rise in immune status of the
human population and globalization.

Risk assessment includes risk identification, analysis, and

evaluation. It involves identification of hazards and the evaluation
of risk associated with exposure to those hazards. All quality
risk evaluations must be based on scientific and process-specific
knowledge and ultimately linked primarily to patient safety. Risk
assessment is based on the strong understanding of the underlying
science, applicable regulations and related processes. Collectively,
these components are to be assessed first and foremost with respect
to the potential impact to the patient (USP <1111>).

Risk identification is the most critical step for risk assessment.

You cannot analyze, prioritize or communicate what you don’t
identify. If this question is not tackled honestly with the suitable
knowledge base, ignored or avoided, then risk assessment becomes
a futile exercise. Only when the proper risk question is well defined,
through understanding the product’s mode of delivery and how a
contaminated product can harm the patient; then alone can adequate
risk management tools be identified and the types of information that
needs to be collected to address the risk question and risk evaluation
step be initiated. A truthful, well thought through or investigated
answer will also benefit in the choice of knowledge base required to
perform a risk assessment. There are a set of questions which should
be asked to identify the real risk. These questions may not be simple
if the appropriate knowledge base is lacking from the get-go. At
this risk question stage very specific experience may be required to
understand risk to patient.

Clearly defining the risk question also facilitates selection of

the appropriate tool(s), identifies relevant data, information and
assumptions, assists in the identification of resources, responsibilities
and accountabilities. Knowledge base to identify the risk question is
the first step, the second step is communicating the real patient risk
to management and the operational team.
 Paper Compliance vs Factual Contamination Control 53

Labels are widely accepted, such as risk management, quality

management and patient safety, etc. But the question remains, who
is knowledgeable enough to define the risk question? Who has
the clinical background to understand the effects of the types of
contaminations on the patient? What might go wrong? What is the
likelihood (probability) it will go wrong? What are the consequences
(severity)? These questions should not be taken lightly.

A good example for this question would be “What is the risk

to patient via a nasal product?”. Under normal circumstances, the
answer would be, it is a non-sterile product, stringent controls such
as those for aseptic processing are not required. Or, we have space
in the warehouse, we can set up a modular cleanroom. The testing is
not difficult. Per USP <1111> these products should show absence of
Staphylococcus aureus and Pseudomonas aeruginosa. Hence if personnel
and water systems are well controlled there should be no other risks.

Is this the correct answer to the question? It must be noted that

the compendial Microbial Limits Tests are not intended to serve as
a tests for objectionable organisms, reading the fine print under the
tables in USP <1111> clarifies that these are not all the objectionable
organisms and it is the responsibility of the manufacturer to assess
other organisms that could be objectionable in the product. The other
questions normally asked would be if the product supports growth
or does it have adequate antimicrobial preservation? It is important to
note that antimicrobial efficacy testing is performed using Aspergillus
brasiliensis, but as evident by the infection causing mold species, some
may circumvent antimicrobial efficacy.

These questions and assumption are not enough to identify the

risk. Experts in the global health care community argue that fungal
infections are drastically underdiagnosed (Ponikau et al., 1999).

The known, emerging, and re-emerging infections that are

related to bacterial and mold contamination would set the stage for
risk assessment. The most common bacteria isolated from pediatric
and adult patients with sinusitis are Streptococcus pneumoniae,
54 Cleanroom Contamination Prevention and Control

Haemophilus influenzae, Moraxella catarrhalis, and Streptococcus pyogenes.

Staphylococcus aureus and anaerobic bacteria such as Prevotella and
Porphyromonas, Fusobacterium and Peptostreptococcus species are the
main isolates in chronic sinusitis. Pseudomonas aeruginosa and other
aerobic and facultative Gram-negative rods are commonly isolated
from patients with sinusitis and immunocompromised patients.

Chronic invasive fungal rhinosinusitis shows fungal hyphae

infiltrating mucosa, blood vessels, or bone. These include but are not
limited to Bipolaris, Curvularia, Alternaria and Aspergillus species, as
well as various genera and species belonging to Zygomycetes. Sinus
infections can be categorized as non-invasive and invasive, are hard
to treat and are on the rise.

Non-invasive fungal sinusitis, otherwise called mycetoma or

fungus ball is primarily caused by Aspergillus fumigatus, resulting
in chronic rhinosinusitis in predominantly older immunocompetent
(patients that typically have an intact immune system) females.
Other causative agents include dematiaceous (Bipolaris, Curvularia,
and Alternaria) and hyaline molds (Aspergillus and Fusarium).
Depending on the progression of the disease and the immune status
of the host, invasive fungal sinusitis can be categorized into acute
(severe), chronic (persisting for a long time or constantly recurring),
or granulomatous (an inflammatory tumor or growth) subtypes.
Causative agents most commonly identified are Aspergillus and fungi
of the order Murocales (Zygomycetes). Invasive Fungal Sinusitis
may lead to orbital (cavity or socket of the skull in which the eye
and its appendages are situated) involvement and may cause visual
loss. Altered mental status is a concern due to spread of the infection
to the central nervous system (Madsen and Moldenhauer, 2013).

As stated above, the degree of scientific objectivity used when

answering the risk question will dictate what areas should be
evaluated and assessed for risk.

• Facility controls; need for a cleanroom with correct classification

preferably not in the warehouse as the cardboard, pallets, etc. are
 Paper Compliance vs Factual Contamination Control 55

sources of contamination that can be inadvertently brought into

the cleanroom through personnel and material flows (Lungqvist
and Reinmüller, 2002).

• Material controls that will ensure that the raw materials are
controlled for known bacteria and fungi that cause nasal, sinus,
orbital and other infections via the nasal passage.

• Adequate process controls to establish and perform in-process

testing there by ensuring that the contaminants are eliminated
and do not make it into the product.

• Personnel flows by training personnel about the contaminants

they can bring into the cleanroom.

• Cleaning and disinfection to ensure that the facility and

equipment cleaning is adequate and does not allow propagation
and proliferation of the contaminants.

• Monitoring controls to assess contaminants and evaluate their

effect on patients.

• Quality testing to ensure that the contaminants can be recovered

through the testing methods used.

Planned risk assessments conducted in advance enable quality to

be built into facilities and processes and risk can be reduced, while
quality risk management allows assessment, control, communication
and review of risks through the product’s life cycle. Good leadership,
clear communication and organizational culture are the most impor­
tant critical success factors for the implementation of risk assessment
and management practices in the pharmaceutical industry.

There are certain ways that can be used to effectively manage

the risks, these are as follows:

• There should be an implementation of a defined process.

56 Cleanroom Contamination Prevention and Control

• Risks must be identified.

• Objective measures should be defined to quantify risks.
• Risks should be prioritized based on impact and probability.
• Developing and executing mitigation strategy.
• Monitoring the mitigation strategy or effectiveness.
• Providing oversight in order to ensure consistency and
compliance.
• Implementing an active feedback loop.

The evaluation of the risk to quality should be based on scientific

knowledge and ultimately linked to the protection of the patient
(WHO, Annex 2).

Risk evaluation means comparison of the estimated risk to given

risk criteria using a quantitative or qualitative scale to determine the
significance of the risk. Once the risk question is honestly answered
the risk evaluation step ensues. Risk evaluations consider the strength
of evidence for all three of the fundamental questions mentioned
in the beginning. In performing an effective risk assessment, the
validity and robustness of the data collected is important because
it governs the quality of the output. Here again, the uncertainty is
due to combination of incomplete knowledge about a process and
its expected or unexpected variability. Typical sources of uncertainty
include gaps in knowledge, gaps in pharmaceutical science and
process understanding, challenge sources, and probability of problem
detection.

The output of a risk assessment is either a quantitative estimate

of risk or a qualitative description of a range of risk. When risk is
expressed quantitatively, a numerical probability is used. Alter-
natively, risk can be expressed using qualitative descriptors, such as
“high,” “medium,” or “low,” which should be defined in as much
detail as possible. Sometimes a risk score is used to further define
descriptors in risk ranking.
 Paper Compliance vs Factual Contamination Control 57

Risk assessment tool selection is used as a method to organize

data, decide what steps may be necessary to reduce or control risk and
to help make appropriate decisions. The goal of using risk assessment
tools is not to create lengthy and complicated paperwork and identify
what risk number to assign. The tool could be one or a combination of
tools as long it is simple, and the real risks can be addressed.

Risk assessment/management tools such as FMEA (Failure

Modes Effect Analysis), HACCP (Hazard Analysis and Critical
Control Point), HAZOP (Hazard and Operability Analysis) and
others have turned into buzz words without understanding their
applicability to the product, processes or operations. To understand
risk assessment, it is prudent to comprehend the history and the
applicability of risk assessment tools at our disposal.

Fault-Tree Analysis is a method used to identify root causes of

an assumed failure or problem. It can be used to evaluate system or
subsystem failures one at a time but can combine multiple causes of
failure by identifying links between the systems and sub-systems.
This method relies upon full process understanding to comprehend
risk factors. It was initially used in 1962 for the US Air Force by
Bell Telephone Laboratories on the Minuteman Weapon System.
Since then, the technique has been adopted and adapted by many
industries who are interested in reliability engineering in all major
fields of engineering and now widely used in all other industries. The
deductive analysis begins with a general conclusion, then attempts
to determine the specific causes of the conclusion by constructing a
logic diagram called a fault tree. This is also known as taking a top-
down approach.

Hazard and Operability Analysis technique was initially developed

in the 1960s to analyze major chemical process systems but has
since been extended to other areas, including mining operations,
other types of process systems and other complex systems such
as nuclear power plants and software development. It is now
becoming a popular tool for risk assessment in medical products.
It is a structured and systematic technique for system examination
58 Cleanroom Contamination Prevention and Control

and risk management. HAZOP is often used as a technique for

identifying potential hazards in a system and identifying operability
problems likely to lead to nonconforming products. HAZOP is
based on a theory that assumes risk events are caused by deviations
from design or operating intentions (WHO, 2007). This approach
in HAZOP methodology helps stimulate the imagination of team
members when exploring potential deviations. As a risk assessment
tool, HAZOP is often described as:

• A brainstorming technique.
• A qualitative risk assessment tool.
• An inductive risk assessment tool, meaning that it is a “bottom-
up” risk identification approach, where success relies on the
ability of subject matter experts (SMEs) to predict deviations
based on past experiences and general subject matter expertise.

Hazard Analysis and Critical Control Point was developed in the late
1950s by a team of food scientists and engineers from The Pillsbury
Company, the Natick Research Laboratories, and the National
Aeronautics and Space Administration. The team developed a system
designed to build quality into the product to ensure food safety
for the manned space program (Stier and Surak, 2008). It proposes
identifying and implementing process controls that consistently
and effectively prevent hazard conditions from occurring. It is a
proactive approach used in the food industry for a long time as the
risk of contamination in food is very high. It purports preventive
controls rather than ability to detect which is a retroactive approach
for risk management (WHO, 2003).

HACCP is based on seven basic principles.

• Conduct a Hazard Analysis.
• Identify the Critical Control Points (CCP).
• Establish Critical Limits.
• Monitor CCP.
 Paper Compliance vs Factual Contamination Control 59
Figure 1 Seven principles of HACCP

• Establish Corrective Action.

• Verification.
• Recordkeeping.

Failure Modes Effect Analysis was developed by the American

Military at the end of the 1940s. Munitions malfunctioning led them
to develop a methodology that would eliminate all the potential root
causes. It proposes comprehensive understanding of the process
and considers establishing critical control points which are defined
prior to initiating the assessment. This helps assess potential failure
modes for processes, and the probable effect on performance of the
product. FMEA is highly dependent upon a strong understanding
or the product, process, facility, etc., leading to a risk score for each
failure mode (Stamatis, 2003; Dyadem, 2003; McDermott et al., 1996;
Zimmermann and Hentschel, 2011). Sometimes FMEA is extended to
FMECA (Failure Mode, Effects, and Criticality Analysis) to indicate
that criticality analysis is also performed.
60 Cleanroom Contamination Prevention and Control
Figure 2 FMEA

There are two broad categories of FMEA, Design FMEA (DFMEA)

and Process FMEA (PFMEA).

Design FMEA explores the possibility of product malfunctions,

reduced product life, safety and regulatory concerns derived from:
• Material properties.
• Geometry.
• Tolerances.
• Interfaces with other components and/or systems.
• Engineering noise: environments, user profile, degradation,
systems interactions.

Process FMEA (PFMEA) discovers failure that impacts product

quality, reduced reliability of the process, customer dissatisfaction,
and safety or environmental hazards derived from:
• Human factors.
 Paper Compliance vs Factual Contamination Control 61

• Methods followed while processing.

• Materials used.
• Machines utilized.
• Measurement systems impact on acceptance.
• Environmental factors on process performance.

Preliminary Hazard Analysis (PHA) is an initial high-level screening

exercise that can be used to identify, describe, and rank major hazards
during the conceptual stage. This technique can also be used to identify
possible consequences such as likelihood of occurrence and provide
recommendations for hazard mitigation following the general HAZID
(Hazard Identification) approach. HAZID is a qualitative technique
for the early identification of potential hazards and threats. PHA does
not take a long time to be conducted and provides information early
in the life cycle of the project (CDER, 2006).

Risk Ranking and Filtering is a common facilitation method

used for risk management. This method is also known as “Relative
Risk Ranking,” “Risk Indexing,” and “Risk Matrix and Filtering.”
Its intent is to provide sharper focus to the critical risks within a
system, typically, from a large and complex set of risk scenarios.
Risk Ranking and Filtering works by breaking down overall risk
into risk components and evaluating those components and their
individual contributions to overall risk (CDER, 2006).

Statistical methods involve carrying out a study including

planning, designing, collecting data, analyzing, drawings, meaningful
interpretation and reporting of the research findings. The statistical
analysis gives meaning to the meaningless numbers, thereby
breathing life into lifeless data. The results and inferences are precise
only if proper statistical tests are used.

Ishikawa Diagram (fishbone diagram) was pioneered by Kaoru

Ishikawa, who contributed to the concept of Quality Management
in Japan. An Ishikawa diagram basically helps in understanding the
62 Cleanroom Contamination Prevention and Control

“cause and effect” relationship for solving a problem. It is a very

helpful tool as it gives a pictorial representation of what is the cause
of a problem or a phenomenon, what factors have a high/low impact
to those problem/phenomena and how can the situation be resolved.
The Ishikawa is drawn like a fishbone and helps a person to “see”
the causes and effects in a relationship (Das et al., 2014).

Figure 3 Ishikawa diagram

Risk assessment can be simple and need not be onerous. Per Whyte
and Eaton, 2004), the microbial risk to aseptically manufactured
products in pharmaceutical cleanrooms can be assessed using
fundamental equations that model the dispersion, transfer and
deposition of microbial contamination, and the use of numerical
values or risk descriptors. This can be done in two-stages; the
first stage is used to assess the transfer of contamination from all
the sources within the cleanroom suite and the second stage is
used to assess both air and surface contact contamination within
critical production areas. These two methods can be used to assess
and reduce microbial risk at the preliminary design stage of the
cleanroom and associated manufacturing process or, retrospectively,
for an established manufacturing operation.

Microbial risk assessment in pharmaceutical cleanrooms can be

performed by the formula below.
 Paper Compliance vs Factual Contamination Control 63

Number of microbes deposited on product equals

C × S × Pd × Pa × T
C = Concentration of microbes in the source.
S = Quantity of air or material dispersed from a source over
time.
Pd = Proportion of organisms effectively transferred.
Pa = Proportion of microorganisms that arrive into product area.
A = Area onto which the organisms are deposited.
T = Time during which microbes could be transferred.

Risk can be further broken down into:

0 = No risk.
0.5 = Very low risk.
1 = Low risk.
1.5 = Medium risk.
2 = High risk.

A different way of looking at risk

Risk from microbial contamination equals A × B × C × D where:

A = Contamination arising from a source.
B = Ease of dispersion or transfer.
C = Proximity of source to critical area or process.
D = Effectiveness of control method (barrier, sealed container,
antimicrobials, etc.)

Risk assessments may be qualitative or quantitative as long as the

true risk to patient is identified and addressed.
64 Cleanroom Contamination Prevention and Control

Qualitative: Qualitative analysis uses subjective information in

determining a conclusion of a given unit without proper statistical
outcome. Qualitative analysis is used for subjective decisions based
on the non-quantifiable information; it is also used during root cause
analysis to come up with possible solutions.

Quantitative: Quantitative analysis is a structured statistical

analysis to estimate the risk and is a combination of Severity,
Probability and Detectability.

Risk Priority Number (RPN) can be established during

quantitative risk analysis and it is calculated by multiplication of
identified values of Severity (S), Probability (P), and Detectability
(D). The RPN rating scale can further determine the level of risk as
low, medium and high. This risk ranking can be used in prioritizing
risk control or mitigation.

Tools and technology

This proposed methodology by Whyte and Eaton of understanding
the source, concentration and transport of contamination can also
be applied to particulate contamination. Additional tools such
as investigational airflow visualization using buoyant media can
greatly benefit in identifying the source; areas of contamination
concentration, movement or transport of contaminants (microbial or
particulate) and reservoirs for contaminants when there are pockets
of dead air. Tools such as threshold detectors equipped with a
laser can be used in conjunction with buoyant smoke and particle
counting to get a precise picture of the behavior of contamination in
the cleanroom and around the open product. The L and R method
as proposed in USP <1116>, is a good example of a modern-day risk
assessment tool for contamination.

In the case of particle contamination, it is crucial to understand

the particle size and the nature of particles. The particle size channel
available in particle counting devices, may not be able to detect
particle contamination which poses a risk; for example, fibers may
 Paper Compliance vs Factual Contamination Control 65

not be detected; while the cleanroom particle counts may be within

acceptable levels, macro particles may be settling on product and
lowering yield, while posing risk to patient. Particle contamination
in drugs as well as devices may be very critical depending upon the
mode of administration.

Static attraction is responsible for a majority of particle contamin-

ation related yield losses in many medical device manufacturing
operations. Devices at risk include widely diverging products
such as catheters, stents, optical lenses, IVs, syringes, hip and knee
replacements, pacemakers, breast implants, and blood filters. As
combination products are part drug, part device, static charge may
affect the function of the device component, leading to risk to patient.
With the advent of robotics for drug manufacturing, static charge is
a new challenge that pharmaceuticals will have to face and resolve.

Without adequate understanding of the product or process and

robust subject matter expertise in specific disciplines, small risks
may receive unwarranted attention, while large ones are neglected.
Hence fussing over risk tools and risk ranking may not lead to
identification or control of pertinent risks. Per WHO Guidance:
“It is not always appropriate nor always necessary to use a formal
risk management process (using recognized tools and/or internal
procedures, e.g. standard operating procedures (SOPs). The use
of an informal risk management process (using empirical tools or
internal procedures) can also be considered acceptable.” The degree
of rigor and formality of quality risk management should reflect
available knowledge and be commensurate with the complexity
and/or criticality of the issue to be addressed.

The key to successful risk assessment is knowledge base and

expert judgement. Judgment involves the weighing of available
evidence and reaching a balanced conclusion from that evidence.
No tool can stand alone, the plan must be inclusive of Good
Manufacturing Practices. All the risk assessment tools have one
thing in common – “knowledge base”.
66 Cleanroom Contamination Prevention and Control

The identification of experts in order to seek adequate

knowledge base requires that one develop some criteria by which
expertise can be measured. Generally, an expert is one who has an
in depth understanding about a specific area or field. An expert is
someone who has succeeded in making decisions and judgements
simpler through knowing what to pay attention to and what to
ignore. The industry is over-saturated with experts; however, the
pros are few and far between. The selection of an SME should
depend upon, credibility, efficiency, and interaction skills. The
expert should also be free from motivational biases caused by
economic, political, or other interest. Experts should be willing to
participate in and be accountable for their judgments. How the
experts are to be organized also impacts the selection; the experts
can be complementary, each bringing unique know-how to the
question. For example, for adequate cleanroom design, experts with
high level knowledge in heating, ventilation, and air conditioning
(HVAC), airflows, particulate, etc., can work cohesively to address
contamination prevention. Here, they act more as a team and should
be selected to cover the disciplines needed.

Risk Control includes decision-making to reduce and/or

accept risks. The purpose of risk control is to reduce the risk to an
acceptable level. The amount of effort used for risk control should
be proportional to the significance of the risk. Often decision makers
might use different processes, including benefit-cost analysis, for
understanding the optimal level of risk control. These are the basic
questions when establishing risk controls.

• Is the risk above an acceptable level?

• What can be done to reduce or eliminate risks?
• What is the appropriate balance among benefits, risks and
resources?
• Are new risks introduced as a result of the identified risks being
controlled?

Risk reduction might include actions taken to mitigate the severity

and probability of harm to patient. Processes that improve the
 Paper Compliance vs Factual Contamination Control 67

detectability of hazard and quality risks might also be used as part

of a risk control strategy. The implementation of risk reduction
measures can introduce new risks into the system or increase the
significance of already existing risks. Hence, it might be appropriate
to revisit the risk assessment to identify and evaluate any possible
change in risk after implementing a risk reduction process.

Risk acceptance can be a formal decision to accept the residual

risk or it can be a passive decision in which residual risks are not
specified. For some types of harms, even the best controls might not
eliminate risk. This is often the case in retroactive risk assessment.

Revisiting the nasal product case study, a credible SME would

be able to identify contamination risk due to utilization of a
modular cleanroom inside a warehouse which is laden with mold.
Risk control measures may include using a barrier such as open or
closed Restricted Access Barrier Systems (RABS), which may create
new risks as the barrier may not have its own air supply due to the
cleanroom being modular. The risk of contamination does not only
depend upon the concentration of contaminants, which is of critical
importance, but also on the motion of contaminants (Ljungqvist and
Reinmüller, 2002). The question arises; can contamination from the
surrounding cleanroom be introduced into the RABS and can the
contamination entering the barrier get introduced into the product?
If personnel and material flow remain unchanged, mold may be
carried into the barrier.

The other scenario may be to use a contract manufacturing

facility, or look to build a cleanroom with adequate controls, but not
in the warehouse.

If this risk is not identified upfront and the nasal product is treated
as any other non-sterile product, the result for the manufacturer
may be intermittent contaminated lots. Once the controls have been
defined, it is worth asking the following questions.

• Does the control measure modify the risk?

68 Cleanroom Contamination Prevention and Control

• By addressing one risk, was a different risk created?

Some controls may reduce one aspect of the risk while increasing
another! Controls may include changes to facilities, processes,
policies, procedures, practices, etc.

Controls are usually categorized as either Preventive, Detective

or Reactive. This is based primarily on where in a risk’s lifecycle do
they apply and as a result, do they modify the likelihood and or the
impact of the risk. Preventive controls apply in the initial stages of
a product’s lifecycle, as exemplified in the nasal product case study.
They primarily reduce the likelihood of the risk occurring. Detective
controls usually apply somewhere in the middle of the risk’s life.
Detective controls that are early in the risk’s life usually prevent
likelihood of disastrous scenarios.

Reactive controls apply towards the end of a risk’s life when the
impact is imminent or being felt. They are focused on modifying
impact; and the impact could be possible harm to patient or
regulatory scrutiny. During the Risk Control stage, decisions are
made on which risks, if any, require mitigation and the necessary
actions to be taken in order to reduce or avoid all prioritized risks,
as appropriate and practical.

Risk Communication is sharing of information about risk and risk

management between decision makers and other stakeholders. All
parties should communicate at all stages of the risk management
process. Often communications might include those within the
company and may involve regulatory authorities. The information
must relate to the existence, nature, form, probability, severity,
acceptability, control, treatment, detectability, or other aspects of
risks to quality.

It is a common understanding that risk is defined as the com-

bination of the probability of occurrence of harm and the severity
of that harm. However, achieving a shared understanding of the
application of risk management among diverse stakeholders is
 Paper Compliance vs Factual Contamination Control 69

difficult because each stakeholder might perceive different potential

harms, place a different probability on each harm occurring and
attribute different severities to each harm (CDER, 2006). In relation
to pharmaceuticals, although there are a variety of stakeholders,
including patients and medical practitioners as well as government
and industry, the protection of the patient by managing the risk to
quality should be considered of prime importance.

Risk Review is the mechanism to review or monitor events. The

output or results of the risk management process should be reviewed
to take into account new knowledge and experience gained during
risk assessment. Once a quality risk management process has been
initiated, that process should continue to be utilized for events that
might impact the original quality risk management decision, whether
these events are planned (e.g., results of product review, inspections,
audits, change control) or unplanned (e.g., root cause from failure
investigations). The frequency of any review should be based upon
the level of risk. Risk review might include reconsideration of risk
acceptance decisions.

MANAGING RISK
Risk can only be effectively managed when it is identified correctly,
assessed cohesively, considered for further mitigation and
communicated to all stakeholders. Risk managed in one area and
ignored in another beats the purpose of risk assessment as each area
is the sum total of the big picture – which is risk to patient.

Deterrents to risk assessments

• Risk assessments take resources as there are batches to be
released. It is hard to make the business case to invest more
resources on risk management when we cannot measure the
results. The key question is how do you measure the avoided
costs from an improved risk assessment?
70 Cleanroom Contamination Prevention and Control

• Risk assessments lead to major improvements such as new

technologies – there is no guarantee those will work.

• So far, we have not been hit with any major surprises when it
comes to risk. Why change?

Common pitfalls during risk assessment

• Retroactive risk assessment when product or process fails or
when cited by regulators.
• Product’s impact on patient not understood.
• Not knowing how or where to start.
• Might miss a failure mode or an effect outside the experiences of
the company, gap in knowledge and experience.
• No agreement on use of tools.
• Not starting in the design stage.
• Taking on too large a scope instead of breaking down into parts
and then connecting the dots.
• Prioritizing may be incorrect during assessment and control.
• Risk assessment project mismanaged, if the team forgets to
document, an important failure mode could be left alone, waiting
to occur.
• Time wasted on things that do not matter.
• Not including operators; operators can have good information
on failure modes and on the effectiveness of control systems.
• Ignoring risk from suppliers; paper audits by the quality
department do not assess risk, technical audits with SMEs can
expose risks.
 Paper Compliance vs Factual Contamination Control 71

• Not getting into the details, a superficial look at the process will
miss many failure modes.
• Not assessing each product, bracketing or ignoring product types
or manufacturing lines. Often all sterile products (aseptically
manufactured, terminally sterilized, oncology products, etc.)
will be lumped into one risk assessment. Processes and controls
may be starkly different.
• Use of risk assessment templates; every product or process is
not identical.
• Templates transferred from company to company by personnel.
• Justifying controls; optimism in assuming controls are better
than they are.
• Assuming detection controls apply when they don’t
• Poor user adoption, getting team members to actually follow a
process, use the tools and stick to the methodology.
• Unrealized benefits, risks can minimize a project’s benefits
overnight, or they could be slowly degraded through inefficient
management practices.
• Late-running projects, unforeseen risks can significantly slow
down a project because it takes time to understand them, analyze
them and prepare management plans to monitor, act on and track
them.
• Overspent budgets; budget overruns happen when risks and
the associated actions related to managing them effectively are
not budgeted for.
• Failure; the worst case scenario for failing to adequately manage
risk. Risk assessment is never completed or never produces
anything of value.
• Sub-optimal actions are taken to treat the risk.
72 Cleanroom Contamination Prevention and Control

CONCLUSION
It is commonly understood that risk is defined as the combination of
the probability of occurrence of harm and the severity of that harm.
However, achieving a shared understanding of the application of
risk management among diverse stakeholders is difficult because
each stakeholder might perceive different potential harms, place a
different probability on each harm occurring and attribute different
severities to each harm (CDER, 2006).

ICH Q9, Quality Risk Management provides an excellent high-

level framework for the use of risk management in pharmaceutical
product development and manufacturing quality decision-making
applications. It is a landmark document in acknowledging risk
management as a standard and acceptable quality system practice
to facilitate good decision-making with regard to risk identification,
resource prioritization, and risk mitigation/elimination, as appropriate
(Frank et al., 2008).

Formal risk assessments sometimes fail to add value or clarity

to a situation because risk assessments often address root cause
analysis superficially, resulting in ineffective risk-control actions.
In addition, because risk assessments are often performed by
busy people, the results are often not supported by scientific rigor
as they should be. This can lead to high levels of subjectivity and
uncertainty in outputs and conclusions, which further contribute to
risk. Additionally, in-house expertise may be subjective or may lack
the experience to make value judgements. Risk assessment cannot
be a part time exercise and cannot be rushed. Risk management is
for long-term strategic thinking (CDER, 2006).

Too often, management teams are overconfident in their

knowledge of what’s going on in the organization or over-rely on
annual financial audit results, and they do not realistically assess the
chance of a risk event occurring. Companies roll the dice, and they
may be fine until something happens, and the stakeholders as well
as regulators will hold management responsible. Then comes the
major turmoil and turnover in top positions. The strategic planning
 Paper Compliance vs Factual Contamination Control 73

process is parochial at many organizations: top-down, numbers-

driven, and lacking an appropriate level of risk awareness; this is
shortsightedness on the part of management. Risk assessment and
management might not be on the mind of managers; it may be about
production and expansion to meet the company’s or personal goals.
Clearly, risk management is interlaced with how leadership thinks.
This may stem from lack of understanding about the different types
of risks; compliance, strategic, operational, preventable, treatable,
inherent and patient.

Without clear understanding, ownership and accountability for

risk management, everyone in the organization assumes someone
else is taking care of it.

The flip side of risk is opportunity to improve. A risk management

culture actually presents organizations with opportunities as risk
management is not limiting or constraining. A risk management
culture enables an organization to be more nimble, adaptable, and
change ready. Thinking about risk management as a friend and
not a foe makes obstacles clearer and gives early warnings which
can help make timely course modifications. If an organization has
the culture of working in silos; talking, let alone working, across
functions is difficult; everyone in an organization needs to own
risk management. In fact, involving staff enterprise-wide in risk
assessment/management efforts is one way to break down those silos.

Circumstances change, business evolves, processes change,

technology changes. In order to get the most out of risk assessments,
it is important to think one step ahead; risk assessments should be
dynamic. They should not just report on historical risk, but also
where there is risk currently and where it may be in the future. By
looking forward, risk management should reflect the most likely
scenario of how the risk will impact the company as the company
grows, adds new products or expands capacity.

Per Pharmaceutical CGMPs for the 21st Century – A Risk-Based

Approach (FDA, 2004), efficient risk management requires using the
74 Cleanroom Contamination Prevention and Control

best scientific data, developing quality standards, and using efficient

systems and practices that provide clear and consistent decisions and
communications for the American public and regulated industry.

REFERENCES
CDER (2006) US Department of Health and Human Services Food and
Drug Administration Center for Drug Evaluation and Research
(CDER) Center for Biologics Evaluation and Research (CBER)
ICH Guidance for Industry Q9 Quality Risk Management Food
and Drug Administration, Rockville, MD http://www.fda.gov/
cber/guidelines.htm

Das, A., Kadwey, P., Mishra, J.K., Moorkoth, S. (2014) Quality Risk
Management (QRM) in Pharmaceutical Industry: Tools and
Methodology International Journal of Pharmaceutical Quality
Assurance 5(3).

Dyadem Press (2003) Guidelines for Failure Modes and Effects Analysis
(FMEA) for Medical Devices. Ontario, Canada.

Frank, T. et al. (2008) Quality risk management principles and

industry case studies. Sponsored by the Product Quality
Research Institute Manufacturing Technology Committee
(PQRI-MTC) (http://www.pqri.org).

Ljungqvist, B., Reinmüller, B. (2002) Clean Room Design: Minimizing

Contamination Through Proper Design. Boca Raton, FL: CRC Press.

Madsen, R.E., Moldenhauer, J. (2013) Contamination Control in

Healthcare Product Manufacturing. Vol. 1. PDA/DHI Publishing,
Bethesda, MD.

McDermott, R. et al. (1996) The Basics of FMEA. Portland, OR.

 Paper Compliance vs Factual Contamination Control 75

Ponikau, J.U., Sherris, D.A., Kern, E.B. et al. (1999) The diagnosis
and incidence of allergic fungal sinusitis. Mayo Clinic Proceedings
74(9): 877–884.

Stamatis, D.H. (2003) Failure Mode and Effect Analysis. FMEA from
Theory to Execution. 2nd ed. Milwaukee, American Society for
Quality, Quality Press.

Stier, R.F., Surak, J.G. (2008) Evolution of HACCP: A Natural

Progression to ISO 22000. Process Control.

US Food and Drug Administration, Center for Drug Evaluation and

Research (FDA CDER) (September 2004) Pharmaceutical cGMPs
for the 21st century – A risk-based approach. (http:// www.fda.
gov/Drugs/default.htm).

United States Pharmacopeial Convention (USP) (2012) USP<1116>,

USP Microbiological Control and Monitoring of Aseptic
Processing Environments. USP 35 1L 697–707.

USP <1111> (2019) USP42-NF37 2S Microbiological Examination of

Nonsterile Products: Acceptance Criteria for Pharmaceutical
Preparations and Substances for Pharmaceutical Use.

World Health Organization (2011) (http://www.who.int/medicines/

areas/quality_safety/quality_assurance/guidelines/en/index.html).

WHO (2007) IEC 61882 – Hazard operability analysis (HAZOP).

Quality Assurance of Pharmaceuticals. A Compendium of
Guidelines and Related Materials. World Health Organization,
Geneva, Switzerland.

WHO (2003) Application of hazard analysis and critical control point

(HACCP) methodology to pharmaceuticals. WHO Technical
Report Series, No. 908, Annex 7. World Health Organization,
Geneva, Switzerland.
76 Cleanroom Contamination Prevention and Control

WHO Guidelines On Quality Risk Management. Annex 2. https://

www.who.int/medicines/areas/quality_safety/quality_assurance/
QualityRiskManagement_QAS10-376Rev2_27082012.pdf Accessed
June 15, 2020.

Whyte, W., Eaton, T. (2004) Microbial risk assessment in pharma-

ceutical cleanrooms. European Journal of Parenteral & Pharmaceutical
Sciences 9(1): 16–23.

Yu, L.X., Amidon, G. Khan, M.A., Hoag, S.W., Polli, J., Raju, G.K.,
Woodcock, J. (2014) Understanding Pharmaceutical Quality by
Design. AAPS Journal 16(4): 771–783.

Zimmermann, H.F., Hentschel, N. (2011) Proposal on How to

Conduct a Biopharmaceutical Process Failure Mode and Effect
Analysis (FMEA) as a Risk Assessment Tool. PDA Journal of
Pharmaceutical Science and Technology 65(5): 506–512.

ABOUT THE AUTHOR

Ziva Abraham has over 35 years of academic, research, clinical and
industrial experience in Microbiology, and Quality Assurance. Ziva
has received her master’s degree in Microbiology and has conducted
research on developing Microbial Insecticides using entomogenous
bacteria and fungi towards her Ph.D. degree.

She worked as a clinical scientist for many years and established

clinical laboratory systems in Israel. In this capacity Ziva evaluated
the first automated microbial identification system, introduced
new technologies for HIV testing, and many rapid testing methods
into the group of laboratories she helped establish and manage for
Maccabi Medical. These laboratories conducted clinical testing in
microbiology, chemistry, parasitology, hematology and immunology.
 Paper Compliance vs Factual Contamination Control 77

She is a passionate microbiologist and mycologist and provides

training worldwide on various microbiology and contamination
control topics. Her clinical experience helps her evaluate and
teach the clinical implications of microbial contamination on
patients. Ziva is passionate about fungi; as in her research she had
collected, identified, and tested hundreds of species. She routinely
teaches hands-on fungal identification as well as investigation
and remediation of mold contamination and most importantly the
emergence of new fungal infections.

Ziva established Microrite, a California based consulting firm,

in 1998 as an independent microbiology consultant. Ziva used her
extensive research experience with fungi and clinical organisms
and their impact on human health to build an internationally
renowned contamination control team of experts. Her team consists
of facilities, cleanroom, airflow, static charge, validation, quality and
microbiology professionals with practical knowledge in their field
and involvement in industry standards and guidance organizations.
Ziva’s team works congruently to resolve client issues, prevent
contamination through design and help clients produce safe
products for human and veterinary use.
 3

DESIGN BASICS OF
PHARMACEUTICAL CLEANROOMS

Wei Sun
Engsysco, Inc.
Farmington Hills, MI
USA

In general, cleanrooms are defined as areas in which particle concen­

tration and environmental conditions are controlled at or within
specified limits. Design of cleanrooms and clean spaces covers
much more than traditional control of air temperature and humidity
as typically for commercial spaces, it may involve the controls of
particle, microbial, electrostatic discharge (ESD), molecular, and
gaseous contamination, airflow distributions and patterns, room
air pressurization, sound and vibration, environmental health,
life safety, industrial engineering aspects, process impacts, and
manufacturing equipment layouts. The objective of good cleanroom
design is to maintain effective contamination control while en­
suring required levels of reliability, productivity, installation, and
operational costs.

Preparations of pharmaceutical, biological, and medical products

require cleanrooms to control viable (living) and nonviable particles
that could impact product sterility. Containment and control of air­
borne viable and nonviable particles inside cleanroom consumes

79
80 Cleanroom Contamination Prevention and Control

significant energy, the design of pharmaceutical cleanrooms should

also conserve energy while to achieve cleanroom contamination
control performances.

It is particularly important to determine critical parameters

with quality assurance to set limits and safety factors for
particle/microbial concentration levels, temperature, humidity,
room pressure, and other control requirements. If not properly
considered during an early stage of design, critical variables to be
controlled for room environment and types of controls could vary
greatly from end users’ intended purposes.

DESIGN CONSIDERATIONS FOR

PHARMACEUTICAL CLEANROOMS
Food and Drug Administration (FDA) CGMP regulations, Code of
Federal Regulations (CFR) Title 21 “Current Good Manufacturing
Practice for Finished Pharmaceuticals” (FDA, 2019) on buildings
and facilities require buildings used in the manufacture, processing,
packing, or holding of a drug product shall be of suitable size,
construction and location to facilitate cleaning, maintenance, and
proper operations. Besides, the flow of components, drug product
containers, closures, labeling, in-process materials, and drug
products through the building or buildings shall be designed to
prevent contamination. Operations shall be performed within
specifically defined areas of adequate size. There shall be separate or
defined areas or such other control systems for the firm’s operations
as are necessary to prevent contamination or mix-ups during the
course.

Functional requirement specifications (FRS), critical parameters

and acceptance criteria, design qualification (DQ), installation
qualification (IQ), operational qualification (OQ), and performance
qualification (PQ) in the cleanroom suites are all required to ensure
proper process performance and validation.
 Design Basics of Pharmaceutical Cleanrooms 81

DQ, IQ, OQ, and PQ protocols, in part, set the acceptance

criteria and control limits for critical environmental parameters such
as temperature, humidity, room pressurization, air change rates,
and operating particle and microbial counts (or air International
Organization for Standardization (ISO) classifications and Good
Manufacturing Practice (GMP) grades). These protocols must
receive defined discipline approvals in compliance with the
owner’s quality policies. The qualification plan must also address
master document updates, Standard Operating Procedures (SOPs),
preventive maintenance (PM), and operator and maintenance
personnel training.

The quality of pharmaceutical products depends on the proper

establishment of critical validation parameters and protocols.
This ensures that the pharmaceutical manufacturing operations
are executed properly and consistently, and maintained such that
any deviations from the critical control parameters are identified,
addressed, and mitigated.

The pharmaceutical process must remain under specific control

throughout the entire product lifecycle, so it is important for design
engineers to avoid including tangential or non-process-impacting
parameters on the list of validated parameters.

PIPING AND INSTRUMENTATION DIAGRAMS

One key element of technical design process is the P&ID diagrams
(Piping and Instrumentation Diagrams) which describe the relation­
ships between process equipment, utility systems, and control
instrumentation. It is critical to document the physical sequence
of equipment and systems throughout the design and installation
processes, as well as how these systems interconnect, to ensure drug
product quality and consistency. During design, these diagrams
also provide the basis for developing system control schemes,
process work and material flows, and further safety and operational
investigations, such as the hazard and operability study (HAZOP)
(FDA, 2004).
82 Cleanroom Contamination Prevention and Control

Piping and instrument diagrams are necessary early in the

facility design process to ensure design goals are achieved in
heating, ventilation, and air conditioning (HVAC) system design
(ASHRAE, 2019):

• Room air cleanliness classification typically consists of a facility

room layout plan drawing visually coded to indicate required
pharmaceutical room classifications.

• Flow directions of personnel, material, product and waste.

• Room pressurization settings and resulting air leakage flow

directions.

• Air balance in each room.

• Air handler zoning layout diagrams show the service area of

each air handler system (or subsystem) on a plan view of the
facility room layout. This diagram is used to optimize HVAC
system layouts to minimize cross contamination issues, and to
enhance facility operational responses to equipment failure and
maintenance service outages. It is often necessary to segregate
the exhaust and return HVAC system paths from other HVAC
systems to prevent cross contamination.

System flow and room pressurization diagrams are used throughout

the facility design process, and can be used as the basis of continuous
quality control of cleanroom environmental parameters. It is critical
to develop HVAC system layouts in conjunction with environmental
quality requirements (room classifications) to minimize process
contamination risks, promote stable facility pressurization strategies,
and minimize facility operational challenges during equipment
servicing.

Biomanufacturing and pharmaceutical aseptic clean spaces are

typically arranged in operational suites based on specific process
and formulation requirements. For example, common convention
 Design Basics of Pharmaceutical Cleanrooms 83

positions an aseptic core ISO Class 5 filling area in the innermost

room, which is at the highest pressure, surrounded by areas of
descending pressure and increasing particulate classes and bacterial
levels (see Figure 1).

Figure 1 Example of room pressure settings for an aseptic filling suite

Courtesy of Morgan Polen

In aseptic processing facilities, the area of highest cleanliness is

intentionally placed with lower-cleanliness areas surrounding it,
separated by airlocks and room pressure differentials. A positive
pressure difference of 0.04 to 0.06 inches of water (10 to 15 Pa)
between air cleanliness classifications is common, with the higher-
cleanliness space having the higher pressure. Lower pressure
differences may be acceptable if they are proven effective. A pressure
differential is generally accepted as good manufacturing practice to
inhibit particles from entering a clean suite.
84 Cleanroom Contamination Prevention and Control

PARTICLE SOURCES AND CONTROL METHODS

Although airborne particles and viable organisms may be
minimized by dilution with high air change rates and by supplying
filtered air, the most effective control is to minimize release of these
contaminants in the space. Personnel and machinery are the most
common sources of contamination, and can be isolated from the
product by gowning, masks, and isolation barriers. Careful study
of how each space operates should reveal the most probable sources
of contaminants and help the HVAC designer determine dilution air
quantities and locate supply air outlets and return air inlets. Avoid
duct liners and silencers in supply air ductwork where contaminants
can collect and bacterial and mold spores can accumulate. Ensure
special attention is paid to cleaning and degreasing of metal sheeting
and air ductwork before installation. Ensure the cleaning agent will
not cause flaking of galvanized ductwork or leave residual soap.
Factory-wrapped ducts and components with clean installation and
inspection protocols promote cleanroom system cleanliness.

ISO CLASS AND GMP GRADE DETERMINATION

Airborne particle and microbe levels in aseptic processing areas
are limited by government regulations, with lower limits for more
critical spaces. European and FDA particle limits (EC, 2010; ISPE,
2009) are for the space in full operational mode, and can also be used
conservatively as limits for the space at rest.

Facilities complying with US CGMPS for aseptic processing

must meet particle levels with manufacturing under way. (An ex-
ception is aseptic powder processing, in which airborne particulate
levels at powder filling heads will exceed limits.) There should be no
microbial contaminants in the critical-zone airstream, where filling
and other critical activities occur; this area should be ISO Class 5. The
area immediately around the critical zone should be ISO Class 7. If
the critical area is within an isolator, then the area outside the isola-
tor may be ISO Class 8. Less critical support areas can be ISO Class 8.
For more detail on facility design, see FDA requirements.
 Design Basics of Pharmaceutical Cleanrooms 85

According to the FDA, 20 Air Changes per Hour (ACH) is

usually sufficient for ISO Class 8 rooms; ISO Class 7 and 5 areas
require significantly higher air change rates. Facility requirements
for terminally sterilized products are not defined. EU GMP (EC,
2010) also contains require­ments for aseptic processing, and also
addresses terminally sterilized products. Note that many facilities
are constructed to meet both EU and US GMP guidelines.

CONTAMINATION CONTROLS IN
ROOM AIRFLOW PATTERN DESIGN
Airflow patterns or streamlines in cleanrooms are strongly influenced
by air supply and return or exhaust configurations, their flow rates,
personnel and material traffic paths, and process equipment layout.
The first step of good cleanroom design is to select the air pattern
configurations. Final air pattern design selection is influenced by
the layout of process equipment such as isolator, Restricted Access
Barrier Systems (RABS), autoclave, clean workstation, transport cart,
table, etc. Cleanroom airflow patterns can be categorized as either
unidirectional, non-unidirectional or mixed flow.

Airflow patterns for cleanrooms of ISO Class 5 or cleaner are

typically unidirectional, while for cleanrooms of ISO Class 6 or less-
clean classes, non-unidirectional and mixed flow are more typical.
For all three air patterns, if the application is complex, unique, or
critical, during the design phase mock-ups of the cleanroom or CFD
(Computational Fluid Dynamics) simulations may help designers
avoid turbulent zones and countercurrents. Air streamlines can be
made visible in the mock-ups with the use of nitrogen vapor fogs or
neutral-buoyancy smoke. Figures 2(A) and (B) show some examples
of pharmaceutical cleanroom airflow patterns (Sun et al., 2017; IEST,
2015).

Cleanroom airflow pattern design is often associated with

airflow path design in a larger scope when contamination control
and contaminant removal are also requirements. Table 1 lists some
common concerns and typical treatments.
86 Cleanroom Contamination Prevention and Control
Figure 2 Examples of airflow patterns

(A) Mixed flow – airflow inside (B) Unidirectional flow –

filling room with RABS or mini flow inside isolator
environment chamber

Courtesy of Wei Sun

Table 1 Contamination control treatments

Common concern Typical treatment Airflow path sequence

Product protection Keep personnel and process HEPA-filtered clean air for
from particle downstream of product and • product
contamination by away from up-stream • process area
personnel or process • personnel
Personnel protection Position personnel upstream HEPA-filtered clean air for
from process and provide segregation to • personnel
associated with harmful allow personnel to have • process area inside isolator
contaminants indirect contact with process or glove box
when needed • process exhaust
Remove harmful Provide exhaust air to capture HEPA-filtered clean air for
contaminants from and remove contaminated air • personnel
cleanroom to protect by particles, gases, chemical • process area
room occupants fumes, or microbes, and use • room exhaust
filtered clean air supplied to
the room as makeup air to
offset the exhaust air

Courtesy of Wei Sun

 Design Basics of Pharmaceutical Cleanrooms 87

AIRFLOW INTENSITY DETERMINATION

Table 2 lists typical airflow ranges for various cleanroom classific­
ations. It should be cautioned, though, that the flow or velocity
ranges listed in this table are experience based or opinion based
from the experts writing the cited guidelines. Therefore, design
engineers should use their own judgment based on the actual site
and operational conditions to determine the final airflow rates for
their cleanroom designs.

Table 2 Historical guidance on cleanroom’s air change rates

ISPE Good Practice Guide: HVAC (2009)
ISO 14 644- 1 Class (ISO 2015)

FDA Aseptic Guidance (2004)

IEST- RP-12.1 RP-1 2.2 (1998)

IEST-IE ST- RP-12.2 (2007)

EU Guidelines to GMP

IEST RP-12.3** (2015)

WHO (2011)

PIC/S (2017)
USP (2004)
FS 209*

ISO Class
20 – – 20 – – 5–48 2–20 2–20
Class 8 100,000

ISO Class 6-20 for

>20 – – 50 – 60–90 20–200 20–200
Class 7 10,000 Grade D

ISO Class 20–40 for

– – – – 150–240 >200 >200
Class 6 1,000 Grade C

Velocity: Velocity: Velocity: 40–60 for Velocity:

39–98 71–106 71–106 Grade B; 71–106
ISO fpm fpm fpm Grade A fpm
Class 100 >100 240–480 > 200 <200
Class 5 (0.2–0.5 (0.36–0.54 (0.36– is based (0.36–
m/s) m/s) 0.54 on 0.54
m/s) velocity m/s)

ISO
Class 10 – – – – – – 300–540
Class 4
Velocity
ISO Velocity
Class 1 – – – – – – 360–540 40–100
Class 3 40–100
fpm
fpm
ISO Class (0.2–0.5
– – – – – – 360–600 (0.2–0.5
Class 2 0.1– m/s)
m/s)
ISO Class
– – – – – – 360–600
Class 1 0.01

• All editions of FS209, A through E(GSA 1966, 1973, 1987, 1988, 1992).
•• Institute of Environmental Sciences and Technology (/EST) changed guidance in 2015 edition to “rule of thumb.”

Courtesy of Wei Sun

88 Cleanroom Contamination Prevention and Control

For decades cleanroom engineers traditionally have used these rules-

of-thumb values for designs. This table approach, however, uses
only the required room cleanliness class to determine an air change
rate (ACR) value and ignores many variables that could significantly
impact the room particle concentration and the required air change
rate, such as particle generations inside the room, efficiencies of high-
efficiency particulate air (HEPA) filters and filters installed in air
handling unit (AHU) and return air fans (RFUs), out-door air intake
particle concentration, particle surface deposition, particle entry
through supply filtered air, particle exit through return air, exhaust
air, and leakage air (particle loss or gain) under pressurization or
depressurization, etc. Intuitively, activities generating higher dust
levels require higher ACRs to dilute particle concentrations than
those generating dust at lower levels. Each cleanroom is unique – its
room cleanliness requirements, space configurations, production or
process activities, HVAC systems, building construction, location,
etc., can impact the ACR requirement for each room. Using the table
approach without considering all these variables could result in
under-designed HVAC systems or cause energy waste (Naughton,
2016; Sun et al., 2017; IEST, 2015; ISPE, 2009).

HVAC SYSTEM CONFIGURATIONS

The HVAC system configuration for commercial or general-purpose
industrial buildings is mainly designed to meet the indoor space’s
heating and cooling loads, or more specifically to achieve the space’s
temperature and humidity requirements. For cleanroom facilities,
however, the HVAC configuration should be prepared such that
it not only meets heating and cooling loads but also satisfies space
air cleanliness requirements with the very same HVAC system.
Satisfying air cleanliness has been traditionally accomplished by
using high airflow rates to dilute a cleanroom’s airborne particle
concentration, which represents the room’s air cleanliness. The
challenge is how to configure a HVAC system when the airflow rate
required by dilution is significantly higher than that required by
heating and cooling loads.
 Design Basics of Pharmaceutical Cleanrooms 89

Figures 3A–C illustrate the typical HVAC configurations of single

primary AHU, primary RFU with secondary AHU and primary fan
filter units (FFUs) with secondary AHU. Engineers can first calculate
the airflow rate required to meet the heating/cooling load and
then calculate the airflow rate required to achieve air cleanliness.
Then the design engineer can select a configuration with necessary
modifications (such as dual return paths) to achieve desired indoor
conditions. Airflow streams can be either mixed, diverted, or a
sequential combination of both.

Figure 3 Basic cleanroom HVAC system configurations

(Sun et al., 2017; IEST, 2015)

(A) Single AHU (Type 1)

(B) Primary RFU with secondary AHU (Type 2)

90 Cleanroom Contamination Prevention and Control
(C) Primary FFUs with secondary AHU (Type 3)

Courtesy of Wei Sun

Instead of using guesswork for selection of an HVAC configuration,

airstream properties (dry and wet bulb temperatures, moisture
content, flow rate, enthalpy, etc.) need to be calculated and psycho­
metric characteristics can be analyzed to obtain more predictable
indoor conditions through computer simulations.

ENERGY SAVING MEASURES TO LOWER

CLEANROOM AIR CHANGE RATES
To maintain the same ISO class in a cleanroom, many options are
available than using a high air change rate alone. When applicable,
the following measures can be used to lower the air change rate or
air velocity requirement (Sun, 2003; 2017):

Select equipment, machinery, furniture, and

room construction materials with lower particle
generation levels
It is intuitive that activities generating higher levels of dust require
higher ACH to dilute particle concentration than those generating
lower levels of dust. Internal particle/dust generation rate could be
 Design Basics of Pharmaceutical Cleanrooms 91

the most significant impact on airflow rate requirement. Various

ranges of particles can be generated by different items inside
a cleanroom, including people, furniture, machinery, process
equipment, and room enclosure construction materials, among
others. One of the major energy and capital wastes is to ignore or
allow high-level particle generation from these materials or products
to occur in cleanrooms without containment, causing design
engineers to use high-level airflow rates to dilute and decrease air
particle concentration.

Design professionals should select cleanroom furniture,

machinery, equipment, and room enclosure construction materials
at the lowest particle generation levels possible. Manufacturers
should list particle generation rates or similar measurements in their
product specifications if the products are intended for cleanroom
applications.

Isolate and remove high-concentration

particles generated in the cleanroom
Once the equipment, machinery, furniture, and room enclosure
construction materials are selected, local minienvironments and
local exhaust could be added to isolate and remove particles from
the equipment, machinery, or processes identified as the sources of
high-concentration particles.

For personnel particle generation, one cost-effective way of

reducing particle generation is enhanced gowning with better
body coverage and gowning protocols. The goal is to minimize the
particles from major sources being dispersed into the remaining
cleanroom spaces.
92 Cleanroom Contamination Prevention and Control

Enhance surface cleaning protocols to

prevent surface particles from turning
into airborne particles
High velocities of supply air and high change rates are intended to
dilute and wash down the particles, yet no return and exhaust system
can completely remove all particles from a cleanroom. Some particles
accumulate on equipment, furniture, and hard-to-reach floor areas
underneath the equipment and furniture. Over time these particles
build up, until a scheduled cleaning and vacuuming removes most
of the residuals. Surface particles that are not removed could reenter
the air and become airborne particles if there is a disturbance on the
exposed surface, which increases the air- borne concentration.

Control particle entry through supply air

Supply air is a mixture of recirculated (return) air and outdoor air.
This mixed air should be filtered by ceiling HEPA or Ultra-Low
Particulate Air (ULPA) filters before it enters a cleanroom. These
filters’ efficiencies determine how many particles could be released
into cleanroom spaces. Higher filter efficiencies reduce the particle
entry through the supply air; however, replacing HEPA filters with
ULPA filters at the room level may not be as cost-effective as using
higher-efficiency filters inside the AHU in terms of air change rate
reduction. Of course, design engineers also need to consider the extra
operating costs from higher pressure drops across higher efficiency
filter media.

Design return and exhaust air systems

effectively for particle exit
Before its reentry as much of the supply air to a cleanroom, the
room return air is drawn through low sidewall grilles or a raised
perforated floor and then recirculated and filtered to remove par­
ticles generated from processes and process equipment. The
particles are removed locally by exhaust air; exhaust devices such
 Design Basics of Pharmaceutical Cleanrooms 93

as direct connections to process equipment, canopies, and hoods

can significantly remove highly concentrated particles. Exhaust air,
unlike return air, typically does not allow reentry. Proper placement
of return or exhaust points close to high-concentration areas can
remove particles more efficiently. Effective return and exhaust
pattern designs should achieve a higher contaminant removal
effectiveness value to ensure fewer particles accumulate on exposed
surfaces and become surface particle contamination in cleanrooms.

Maintain proper pressurization/no depressurization,

which causes particle gain through leakage
If a pressure differential exists, then whenever the door to a clean­
room is opened it is inevitable that air leakage will occur between
the cleanroom enclosure and its surroundings. Particle migrations
following the leakage air paths should be considered, especially
under depressurization and when the air in the surrounding area is
dirtier than the cleanroom. To prevent or minimize particle migration
from surrounding areas, cleanrooms should be kept in relative
positive pressure. Usually, the higher the pressure differential, the
more effective it is in preventing particle migration.

CLEANROOM PRESSURE AND

CONTROL STRATEGIES
Controlling contaminants in cleanrooms requires controlling the
direction of airflow between adjacent spaces that have various levels
of cleanliness classification(s). This is achieved by establishing and
maintaining a pressure differential between the spaces. Air pressure
differences are created mechanically between spaces to introduce
intentional air movement paths through space leakage or openings
(Sun, 2020). Pressurization resists infiltration of unfiltered external
sources of contaminants. It can be achieved by arranging controlled
flow rates of supply, return, and exhaust airstreams to each space:
94 Cleanroom Contamination Prevention and Control

• Positive pressurization
Entering (supply) airflow rate is higher than leaving (exhaust
and/or return) airflow rate in the space.

• Negative pressurization
Entering (supply) airflow rate is lower than leaving (exhaust
and/or return) airflow rate in the space.

A cleanroom envelope (including doors) is a natural barrier to

contain airborne contaminants’ (such as particle, microbial and/or
chemical gas) migration. However, when a door is being opened
for traffic, the initial pressure differential across the door/envelope
disappears much quicker (typically less than 0.25 second) before a
door operation cycle (typical 6–10 seconds) to close the door, also
much quicker than any airflow control devices (such as air valves)
to modulate from prior flow positions to the new positions (1–2
seconds). The magnitude of particle migration is much higher at
the door-in-operation (dynamic) condition than at the door-closed
(static) condition. An effective mechanism to tackle the issue is to
install a two-door airlock with a proper “time delay”, a time delay
between two doors can allow the airlock room air to be fully or
partially replaced by filtered clean air (Sun, 2018). Airlock can reduce
particle migration not only during door operations, but also in door-
closed conditions (Sun, 2018).

ASHRAE published table of Recommended Minimum Pressure

Differential Across Cleanroom Envelope, as shown in Table 3, detail
discussions can be found in ASHRAE Design Guide for Cleanrooms
(Sun, 2020).

An important relationship exists between room pressure and

the “flow differential” of the room, which is the flow rate difference
(ΔV) between the room’s incoming airflow and departing airflow;
this flow differential is typically called the “flow offset.” The sum of
room leakages (ΣQ, both air losses and gains) is the flow offset value
(ΔV) of the room as shown in Figure 4 (Sun, 2003; 2020).
 Design Basics of Pharmaceutical Cleanrooms 95
Table 3 Minimum pressure differential (∆P) requirements
across cleanroom enclosure (Sun et al., 2017; Sun, 2003)

Door in Closed
Door in operation condition (dynamic)
Cleanliness class difference Condition (Static)
between cleanroom and Minimum pressure
adjacent less-clean area differential (∆P) Installation of airlock
between rooms
One-class difference
(e.g., ISO Classes 7 and
0.04 in. (10 Pa) Not required
8 adjacent rooms across
door)
Required if door operation is frequent
(more than 30 times daily)

• Install a two-door airlock to replace

single door which separates two
Two-class difference
areas
(e.g., ISO Classes 6 and
0.04 in. (10 Pa) • Min. 0.02 in. (5 Pa) across each door
8 adjacent rooms across
of the airlock
door)
• Time delay between two doors in
airlock

Not required if door operation is not

frequent (30 times or less daily)
Required

• Install a two-door airlock to replace

Three or More-Class
single door which separates two
Difference (e.g., ISO
0.04 in. (10 Pa) areas
Classes 5 and 8 adjacent
• Min. 0.02 in. (5 Pa) across each door
rooms across door
of the airlock
• Time delay between two doors in
airlock
Courtesy of Wei Sun
96 Cleanroom Contamination Prevention and Control
Figure 4 Room airflow offset (either a surplus or a deficit)
is required to establish pressurization or depressurization

Source: IEST

MULTIPLE-ROOMS (SUITE) UNDER

MULTISTAGE PRESSURIZATIONS
Most pharmaceutical suites consist of multiple rooms which
are often required for various pressures. As shown in Figure 5,
pressurization design for a suite of pressure-controlled rooms is
more complex. In practice, air leakage interactions among rooms
have been neglected in the design industry due to their complexity.
Air loss (air leaking out) from one room could be the air surplus to
surrounding rooms; air gain (air leaking in) to one room could be
the air deficit to surrounding rooms. Air leakage among rooms is
thus in a “network” relationship. Adjusting one room’s offset value
 Design Basics of Pharmaceutical Cleanrooms 97

often affects the adjacent rooms, which might previously have been
balanced for air pressures. This phenomenon is more profound
and intensified in multistage-pressurizations cases. Overlooking
this network effect can cause difficulties in room pressure control,
commissioning and operation. Poorly designed pressurization
could become unpredictable or unstable, or even fail to perform for
contamination control. For detailed procedures, see Sun (2003).

Figure 5 Example of pressure control technique for multiple

rooms in a cleanroom suite with positive and negative pressures

Source: IEST with permission

TEMPERATURE AND RELATIVE HUMIDITY

The United States Pharmacopoeia (USP) limits temperatures to
which finished pharmaceutical products may be exposed to 59 to
86°F (15 to 25°C). The production facility may need tighter limits
than these, based on the owner’s observed product data. Personnel
comfort is also a factor in design. Personnel perspiring in their
protective overgarments can increase particulate and microbial
counts, so lower temperatures and tighter temperature control may
be advantageous.
98 Cleanroom Contamination Prevention and Control

Relative humidity may be critical to the product’s integrity.

Some products are processed or packaged cold and need a low
room dew point to prevent condensation on equipment and vials.
Some products are hygroscopic and require lower humidity than
a condensing coil can provide; in that case, consider desiccant de­
humidification. Caution must be taken in designing low-humidity
(i.e., low-vapor-pressure) spaces to ensure limited moisture
migration through walls and ceilings bordering an unclean space.
Low-humidity spaces should be provided with air locks to reduce
moisture propagation into the low-humidity cleanroom. The im­
portance of positive pressure increases when moisture infiltration
potential becomes an element of the design process. Humidification
is usually needed for personnel comfort but not usually for product
needs; it may also be needed where dust might present an explosion
hazard or where low humidity may hinder handling of dry materials.
Clean steam (free of chemicals and other additives) is preferred for
humidification because it is free of bacteria, but the humidification
system should be free of amines or other contaminants if space air
might contact the product. Humidification control systems often
require careful sensor placement in critical areas and safety shutoff
monitors to prevent over-humidification.

NOISE CONTROL
HVAC noise is a common problem caused by attempts to overcome
the pressure drop of additional air filtration. The noise level
generated must be reduced in lieu of adding duct silencers, which
may harbor bacteria and are difficult to clean. Separate supply and
return fans running at lower tip speeds instead of a single-fan air
handler may reduce generated noise levels. HVAC noise may not be
an issue if production equipment is considerably noisier.
 Design Basics of Pharmaceutical Cleanrooms 99

ENVIRONMENTAL MONITORING AND CONTROLS

Manufacturers must establish control procedures that monitor the
output and validate the performance of manufacturing processes
to prevent variability in the in-process material and the drug
product. Engineering responsibility includes identifying any and
all variables that may foreseeably affect product or process quality,
and assessing these potential problems during quality-driven risk
analysis. Careful identification and control of variables that can
affect product and process quality is necessary to ensure system
performance and compliance. Utility system designs emphasizing
performance stability and consistency through appropriate controls,
alarms, and routine maintenance and inspections are required for
compliance with pharmaceutical regulations. A lack of compensation
for HEPA filter loading is a common HVAC system qualification
challenge; if airflow or pressure controls are not used, appropriate
alternative controls or alarms are required for documentation
of continuous compliance. For most cleanroom applications, a
routine environmental monitoring program verifies that the critical
parameter of room cleanliness is being maintained. For holding
rooms and other specialized applications, ensuring the stability of
HVAC system performance through air filter loading compensation
is usually the most effective way to support consistent facility
operations.

The owner and design engineer must define the tolerable range
of variable value (acceptance criterion) for each critical parameter.
The product’s safety, identity, strength, purity, and quality must
be demonstrated to be unadulterated in that range. The owner
should define action alarm points at the limits of acceptance criteria,
beyond which exposed product may be adulterated. The designer
should select tighter (but achievable) target design values for
critical parameters (in the range of acceptance criteria), along with
appropriate critical parameter monitoring strategies and values for
warning alerts and actionable alarms.
100 Cleanroom Contamination Prevention and Control

Facilities manufacturing penicillin or similar antibiotics

(e.g., cephalosporins) must be physically isolated from other
manufacturing areas and served by a dedicated HVAC system.
Other processes also require dedicated HVAC systems, including
high-potency formulas and formulas that must have dedicated
production facilities.

Facilities manufacturing aseptic/sterile products derived from

chemical synthesis may have different requirements than those
manufacturing biological or biotechnological products. The owner
must define the inspecting agency’s requirements.

AIRLOCKS
Where there are spaces adjoined in series that all have different
cleanliness classifications, a multiple-step pressurization cascade
should be implemented, which should have air flow from the clean­
est spaces to the least clean spaces. Normally, three pressure steps
are used for ISO Class 6, 7, or 8 applications; four pressure steps are
desirable for Class 5 or cleaner applications. Air locks are effective
at minimizing potential particle contamination from surrounding
non-classified or less-clean areas; selection depends on the type of
cleanroom as shown in Figure 6, because some that involve fume or
biological agent operations may have a containment provision. For
biological agent operations, the US Centers for Disease Control and
Prevention (CDC) and National Institutes of Health (NIH) define
four biosafety levels (BSL-1 to BSL-4).

An air lock is a transitional room between adjacent rooms to

prevent airborne cross contamination. Based on relative space
pressure levels, air locks can be classified as follows:

• Cascading
Air lock pressure is between pressures in cleanroom and corridor.

• Bubble
Air lock pressure is above pressures in cleanroom and corridor.
 Design Basics of Pharmaceutical Cleanrooms 101
Figure 6 Air lock types and applications

Source: Sun (2018)

• Sink
Air lock pressure is below pressures in cleanroom and corridor.

• Dual-compartment
A bubble and a sink air lock are connected.

Double-door air locks are often used at cleanroom entrances and

exits. A required time delay (RTD) needs to be specified between
door openings, so both are not open simultaneously, to minimize
possible contamination opportunities. The RTD should be long
enough for HEPA-filtered clean supply air to partially or fully replace
the entire air volume of the air lock room at least once before the
second door is allowed to open. RTD operational procedures often
use hard interlocks (i.e., the second door cannot be opened until
after the required time delay) or soft interlocks, in which procedures
are supplemented by lights or alarms.
102 Cleanroom Contamination Prevention and Control

BARRIER TECHNOLOGY
Cleanrooms designed to meet ISO Class 5 or better require sophis­
ticated technology, considerable equipment, space, and con­trol
systems. Operating such cleanrooms is expensive. Cleanrooms
typically need gowned operators inside to manipulate product
and adjust machinery. Because the operator can be a major source
of particle generation and contamination, it is better to separate
the operator from the controlled environment; this allows the
volume of the controlled space to be reduced significantly to a point
where only the process equipment is enclosed. Using such a separ­
ative device can substantially reduce capital and operating costs
while meeting required airflow patterns and cleanliness levels (ISPE,
2009; ISO, 2004). Separative devices, including microenvironments,
glove boxes, isolators, and RABS, are thus becoming increasingly
popular. These systems are also called barrier technology in pharma­
ceutical industries.

Barrier technology systems must be designed to fit the specific

application and can be highly customized to allow the tasks required
to accomplish the process needs. Applications vary widely based on
product, process equipment, and throughput volume. Barrier tech­
nology systems are typically positive-pressure enclosures around
the filling equipment with multiple glove ports for operator access,
constructed of polished stainless steel with clear, rigid view ports.
Isolator chambers are typically fully sealed and highly pressurized
in respect to the surrounding environment and allow leak air only
through “mouse holes” which are the designated passages of vials
in and out of the unit. RABS sections’ top ceiling and side panels
form an “open-bottom enclosure” to allow internal air to leak into
the surrounding environment through the bottom of the RABS units.
Ancillary systems designed to prevent migration of contaminants
are used for passing stoppers, containers, and tools in and out of
the barrier systems. Important design concerns include accessibility,
ergonomics, integration with mating equipment, decontamination
or sterilization/sanitization procedures, access to service equipment,
filter change, filter certification, process validation, and barrier’s
internal environmental control.
 Design Basics of Pharmaceutical Cleanrooms 103

Extra attention must be paid to

• Product filling, vial, and stopper protection.

• Access to the barrier for sterilized stoppers.
• Interface to the vial washing and dehydrogenation devices.
• Sterilizing product path, including pumps and tubing.
• Airflow patterns inside the barrier, especially at critical points.

If a vapor-forming sanitizing agent such as hydrogen peroxide is

to be used as a surface sanitizer, care must be taken to ensure good
circulation and adequate concentration inside the barrier, as well as
removal of residual vapor in the required time frame. In addition,
because many of the sanitizing agents are strong oxidizers, care
must also be taken in selecting construction materials to ensure
compatibility and their ability to absorb and retain or potentially
outgas the sanitizing agent at a later time.

ISOLATORS
Isolators are designed to provide continuous and complete isolation
of the inside of the isolator from the external room environment
(including its operators). Only installed gloves or robotic arms
are used to manipulate the product. Isolators operate as positive-
pressure devices, and use full wall separation and substantial
overpressure to both physically and aerodynamically separate the
interior from the external room environment. An isolator is a de­
contaminated unit, supplied with Class 100 (ISO 5) or higher air
quality, that provides uncompromised, continuous isolation of its
interior from the external environment (e.g., surrounding cleanroom
air and personnel).

Pharmaceutical industry and pharmacy compounding isolators

are used for maintaining sterility of a drug. This type of strict
design and control is important when producing sterile medicines
because consumers receiving injections, surgical irrigates, or other
104 Cleanroom Contamination Prevention and Control

“parenterally” administered drugs are often highly vulnerable

to infection. As a result, contaminated drugs have caused grave
consequences for the consumer. There are three major types of
isolators:

Closed isolator
“Closed isolator” systems exclude external contamination from the
isolator’s interior by accomplishing material transfer via aseptic
connection to auxiliary equipment, rather than use of openings
to the surrounding environment. Closed systems remain sealed
throughout operations.

Open isolator
“Open isolator” systems are designed to allow for the continuous
ingress and/or egress of materials during operations through one or
more openings such as mouseholes. Mouseholes and these openings
are engineered under continuous over-pressure to exclude external
contamination from entering the isolator chamber.

Negative pressure isolator

While the positive pressure isolator is most common, “negative”
pressure devices also exist for very large industrial operations that
handle toxic products. The “negative pressure isolator,” has become
less common and desirable, but is superior to the traditional bio­
safety cabinet which is vulnerable to contamination and can expose
the worker to toxicological hazards if not operated properly.

Isolator systems may also be designed for applications requiring

operator protection from high-potency and cytotoxic compounds,
while maintaining a sterile internal environment. These tend to be
total containment systems with totally contained product transfer
ports. All internal surfaces are sealed from the external environment
or potential operator exposure.
 Design Basics of Pharmaceutical Cleanrooms 105

RESTRICTED ACCESS BARRIER SYSTEMS

RABS are an alternative to a conventional isolator. RABS design and
control is below the isolator in its ability to assure sterility assurance
and containment, but far better than the traditional laminar air
flow hood or “open process” designs that are progressively being
phased-out by the industries. Once the product is inside the RABS
containers, the need for particulate control and minimum air changes
inside the surrounding cleanroom is reduced, depending on the
degree of protection provided by product packaging. The owner
should determine the necessary critical environmental parameters
and acceptance criteria for each space and processing step.

RABS can be utilized in fill–finish areas. The equipment provides

an enclosed environment which reduces the risk of contaminating
the product, containers, closures, and direct contact surfaces. It
provides a better protection than conventional cleanroom operations
by providing a level of separation between operator and product.
Since the equipment is open to the surrounding room, it is commonly
located in an ISO Class 7 or better environment.

RABS structure is typically built on stainless steel frames with

rigid wall panels, internal environment is commonly considered
or designed for ISO Class 5/Grade A with unidirectional supply
air filtered by HEPA filters. RABS are further divided into two
categories: open RABS (oRABS) or closed RABS (cRABS).

Open RABS
An open operation RABS, by definition, provides recognition that
the barrier doors can be opened for operator intervention(s). The
intervention could occur during either set-up or process time. An
oRABS could have two types: active or passive oRABS.

Active oRABS system has a dedicated air handling system which

is completely independent from the cleanroom’s air supply; while
passive oRABS uses the cleanroom’s ceiling supply air downflow
106 Cleanroom Contamination Prevention and Control

with an air overspill to the surrounding environment. For both types

of oRABS, their barrier doors can be opened for operator intervention
which can increase the risk of contamination of aseptic processes,
especially after the bio-decontamination step is complete. Transfer
devices may include closed or aerodynamic protection at the device-
barrier connection location to maintain a closed separation to the
surrounding environment during the transfer procedure. oRABS
are used to incorporate filling line equipment both for aseptic and
potent products processing. This equipment is a practical means
as a containment solution for processes such as, but not limited to,
milling and sieving purposes.

Other systems, such as a nonsterile powder control booth, may

incorporate passive RABS designs. One such design incorporates a
downflow sampling and weighing cubicle. This arrangement takes
advantage of unidirectional airflow to wash particles down and away
from the operator’s breathing zone. Low-wall air returns at the rear
of the cubicle capture fugitive dust. An arrangement of roughing
and final filters allows air to return to the air handlers and back to
the work zone through ceiling-mounted HEPA filters or recirculated
by fan-filter units. Products involving noxious or solvent vapors
require a once-through air passage design.

Closed RABS
The closed RABS is an intermediate solution between isolators
and open RABS. A closed operation RABS provides a higher level
of contamination control because the RABS barrier doors remain
closed from the point of the last bio-decontamination, through initial
set-up through processing. These systems typically use transfer
systems that are similar to isolator type transfer systems that are
closed and dock with the RABS. cRABS also make use of a gaseous
decontamination system. Other materials coming from the outside
environment must undergo autoclaving prior to RABS entry via
aseptic transfer ports. cRABS has glove ports to access all areas of
the enclosure during filling operations.
 Design Basics of Pharmaceutical Cleanrooms 107

cRABs system has a dedicated air handling system that provides

down-flow air that circulates inside physical barriers, together with
the provision of fresh air make up and ducted exhaust systems.
Materials transfer devices are either a fully closed system with
rapid transfer ports, or devices that connect for interface. In either
case doors remain closed to the surrounding environment during
transfer procedures. Closed RABS internally should be designed
for a Grade A environment and the surrounding area should be
designed for Grade B.

Compared with isolators, cRABS are easier for installation,

validation on airflow and classification, however the surrounding
area must maintain Grade B, while for an isolator it can be
downgraded to the less expensive Grade C.

In particular, a RABS that operates only in closed-door mode

after the equipment setup and sporadic disinfection is performed, is
commonly used now and provides substantial risk mitigation. The
cRABS require all processing interventions to be done using gauntlet
gloves attached to the RABS walls. RABS doors are only opened at
the start of an operation to perform equipment setup, and must be
locked thereafter until the conclusion of operations.

Some RABS designs allow for rare door openings in specified

circumstances. Because this “open RABS” allows for a door to be
opened to the surrounding cleanroom during aseptic operations, the
design may have a higher contamination risk than a RABS that is
kept closed. Therefore, “open RABS” must be operated properly to
realize sterility assurance gains.

GLOVEBOXES
Gloveboxes may not offer the separative control provisions of an
isolator or active RABS. Gloveboxes were originally designed
for non-sterile product applications, such as manipulating a toxic
drug and have a long track record for such non-sterile applications.
Such gloveboxes can be very effective in preventing exposure of an
108 Cleanroom Contamination Prevention and Control

operator to a toxic drug. In some cases, they can also be used to

protect a sterile product when the chamber is supplied with HEPA-
filtered unidirectional air.

LAMINAR AIR FLOW UNITS

ISO Class 5 unidirectional hoods are commonly used in process-
critical applications for aseptic processes, consisting of banks of
HEPA filters, integrity-tested to be pinhole-free. In Laminar Air
Flow (LAF) units, air is drawn in from outside the cabinet through
a pre-filter by means of a fan. The fan then pushes the air drawn in
through the HEPA filters into the working chamber, leaving the air
space after filter surface in a unidirectional flow pattern. This can
be a horizontal flow (crossflow) or vertical flow (downflow). HEPA
filter’s face velocity is usually 70 to 90 fpm (0.35 to 0.45 m/s), but the
user should specify velocity and uniformity requirements.

LAF unidirectional camber usually has clear sidewalls (curtains)

to promote downward airflow and prevent entrainment of space
particles into the hood’s zone of protection. Curtains should extend
below the product critical surface and be designed to prevent
accidental disruption of airflow patterns by personnel. Many
production facilities prefer rigid curtains for easier cleaning and
sanitization.

Most LAF units utilized in pharmaceutical industry are capable

of maintaining a better air cleanliness such as ISO 5 environments
when they are placed in a less-clean ISO 7 or ISO 8 environment.
However, one of the key challenges is that the LAF unit often draws
room air inside surrounding cleanroom through its sidewall panels,
and thus could cause airflow patten distortion inside cleanroom if
the cleanroom’s airflow pattern needs to be maintained without
any disruption.
 Design Basics of Pharmaceutical Cleanrooms 109

SURROUNDING CLEANROOM
ENVIRONMENTAL CONTROL
Return openings for space HVAC should be low on the walls,
to promote downward airflow from supply to return, sweeping
contaminants to the floor and away from the product. Room air
quality can be improved greatly by optimizing supply and return
air register locations to route air flows away from cleaner areas.
In larger spaces, internal return air columns may be necessary.
Combining noncontrolled and controlled areas through return or
exhaust air pathways may also lead to operational challenges by
expanding controlled-area boundaries into zones with activities that
may negatively influence process or product integrity.

Aseptic facilities usually require pinhole-scanned (integrity-

tested) HEPA filters on supply air. Many facilities install HEPA filters
in the supply air to non-aseptic production facilities to minimize
cross contamination from other manufacturing areas served by
the HVAC system. To increase the life of terminal HEPA filters in
aseptic facilities, and to minimize the need to rebalance the supply
system because of differential loading of terminal HEPA filters,
many designers install a high-capacity HEPA bank downstream of
the supply air fan, with constant-volume control to compensate for
primary filter pressure changes and any dehumidifier airflow. The
final HEPA filter is usually in a sealed gel frame, ceiling T-bar grid
often uses continuous gasket foam along its frames with spring clips
to lock the ceiling panels down tightly on its grid frame, maintaining
the integrity of the room envelope to ensure that room pressure is
well maintained.

Aseptic product must be protected by pressurizing isolator

chambers in which it is exposed, to about 0.04–0.06 in of water (10–
15 Pa) above the surrounding cleanroom. However, to RABS systems,
since the units’ bottom is open to the surrounding cleanroom, it is
much harder to achieve any significant positive pressure inside
RABS chamber over the surrounding cleanroom.
110 Cleanroom Contamination Prevention and Control

To keep the pressure differential from dropping to zero when a

door is opened, air locks are often used between spaces of different
air pressures, especially at the entrance to the aseptic fill space itself.
Room pressure is a function of a room enclosure’s airtightness and
the offset flow rate which is the volumetric differential between the
supply air volume and departing air (return and exhaust combined)
volume inside the room. Consider all potential openings, slots,
electrical outlets, annular spaces around pipe penetrations, and door
leakage that could affect the amount of air needed to pressurize the
space. Because room offset airflows and space pressure are closely
related, outdoor or makeup air requirements for cleanrooms are often
dictated by space pressures rather than by the number of occupants.
The HVAC system should be able to handle more makeup air than
needed for commissioning, because door seals can deteriorate over
time.

DECONTAMINATION
Before the initial use of a cleanroom or after a shutdown, the clean­
room must be decontaminated or disinfected to ensure bioburden
and particulate levels are at or below acceptable limits. For some
operations, such as compounding of sterile preparations, surface
disinfection is considered adequate. However, larger-scale cGMP
sterile manufacturing operations typically use decontamination
before final occupancy. Cleanrooms for sterile processing should be
designed to accommodate decontamination or disinfection.

Most early large-volume decontamination processes included

using formaldehyde gas generated by heating paraformaldehyde in
a frying pan or spraying with a mild peracetic acid and wiping all
surfaces, which was very labor intensive. Today, most cleanrooms are
decontaminated by using either chlorine dioxide (CD) or hydrogen
peroxide (H2O2). Regardless of the type of decontamination process
used, the cleanroom should accommodate the process. Factors that
should be considered include:
 Design Basics of Pharmaceutical Cleanrooms 111

• Leak-tightness of the cleanroom shell.

• Compatibility of cleanroom finishes to the decontamination

process.

• Ability to remotely control the process and recirculate the gas.

• Maintaining appropriate humidity levels during the decontam-

ination process.

• Evacuation of the gas after decontamination is complete.

Sometimes it is economically feasible to integrate the gas-generating

equipment with the cleanroom air ducts. This decision is dictated
by the intended gassing frequency, or by the need for automated
recovery preparedness following any kind of bio-event. Strategically
placed, air-tight dampers, gas distribution nozzles, a means to
agitate the gas within the cleanroom (or suite of rooms), and exhaust
equipment for evacuation are some of the components necessary
for automated decontamination. As with all decontamination
procedures, protocols must be developed to demonstrate efficacy.

MAINTAINABILITY
Maintainability for accessibility, frequency of services, spare parts,
rapid diagnostics and repair, reliability and facility uptime, etc.,
should be considered during facility design. A facility that considers
this in its design will be much more reliable and should have fewer
operational and regulatory concerns. Many pharmaceutical facilities
have been designed so that routine maintenance can be performed
from outside the facility’s cleanrooms (except for unidirectional and
terminal HEPA filters, which must be tested twice a year).

Consider how much exposure and risk to product and personnel

exist during maintenance (e.g., how to clean the inside of a glovebox
contaminated by a toxic product). Beyond cleanable room surfaces
that must be sanitized, consider whether and how HVAC equipment
may be decontaminated using the owner’s procedures. Determine
112 Cleanroom Contamination Prevention and Control

whether ductwork must be internally cleaned, and how. Quality

of materials is important to reliability, especially where failure can
compromise a critical parameter or operation.

Reduced flow (without shutdown) HVAC system designs

require energy-efficient and redundant components. When incor­
porating redundant components into systems, it is important to
consider how both maintenance and removal/replacement of a
component would be executed; the effect of redundancy is negated
if there is no way to isolate equipment that needs to be replaced.
Aligning HVAC system layouts with facility operational areas
or suites can save significant operating costs and increase plant
availability.

CONTROLS, MONITORS, AND ALARMS

HVAC system for a few pressurized spaces may be statically
balanced if there is a method of maintaining supply airflow volume
to compensate for filter loading to ensure minimum supply, return,
and exhaust air changes. More complex designs may require
dynamic pressure control. Both filling lines with conveyors and
slide gates between rooms where air moves from one room to
another at varying rates usually require active pressure control to
maintain room’s pressures and their relationships at all times. It is
important to avoid multiple pressurization loops controlled from
the same or interrelated parameters, because this can lead to space
pressurization instabilities. Complications can result from fans in
series controlling similar or related properties.

Improved system stability results from controlling to an air­

flow value at the room space level, and to duct air pressure at the
branch or air handler level. Pressure controls should not overreact
to doors opening and closing, because it is virtually impossible to
pressur­ize a space with a door standing open (refer to Sun (2020) for
detailed information). A door switch is often used to send a signal
to space pressure control to avoid overreaction. Architectural layout
may affect dynamic room pressure control. It is a good practice to
 Design Basics of Pharmaceutical Cleanrooms 113

position such spaces away from exterior walls where wind loading
exists and interior corridors, which typically do not have dynamic
pressure control.

If space air humidity must be maintained to tolerances tighter

than what normal comfort cooling can maintain, consider using
active relative humidity control. If a desiccant dehumidifier is
needed, unit operation over its range of flow must not adversely
affect the ability of the HVAC to deliver a constant air supply
volume to the facility.

Monitor and alarm critical parameters to prove they are under

control. Log alarm data and parameter values during excursions.
Logging may range from a local recorder to direct digital control
(DDC) data storage with controlled access. Software source code
should be traceable, with changes to software under the owner’s
control after qualification is complete. Commercial HVAC software
is usually acceptable, but should be verified with regulatory agencies
before detailed design begins. Also, keep complete calibration
records for sensors, alarms, and recorders of critical parameter data.

When establishing alarm set points, consider that for systems

serving regulated industries, alarms (or deviations from operat­
ional parameter (OP) acceptance limits) often require extensive
documentation of the deviation, corrective actions taken, and any
impact on critical processes and/or products. By setting up early
warning alarm points, the operators can identify a system trending
towards an operational deviation point and can intercede before the
system hits its OP limits, precluding the need to prepare deviation
or excursion reports.
114 Cleanroom Contamination Prevention and Control

REFERENCES
ASHRAE (2019) “Clean spaces.” Chapter 19 in ASHRAE Handbook –
HVAC Applications. Atlanta: ASHRAE.

EC (2010) Annex 1, Manufacture of sterile medicinal products. In EU

guidelines to good manufacturing practice – Medicinal products
for human and veterinary use, Vol. 4 of EudraLex – The rules
governing medicinal products in the European Union. Brussels:

Food and Drug Administration (FDA) (2019) FDA Title 21, Part
211. Current Good Manufacturing Practice for Finished
Pharmaceuticals, Subpart C – Buildings and Facilities.

Food and Drug Administration (FDA) (2004) Guidance on Sterile

Drug Products Produced by Aseptic Processing. US Government
Printing Office.

IEST (2015) IEST-RP-CC012.3 Considerations in cleanroom design.

Contamination Control Division Recommended Practice 012.3.
Arlington Heights, IL: Institute of Environmental Sciences and
Technology.

ISO (2004) ISO 14644 Part 7: Separative devices (clean air hoods,
gloveboxes, isolators and mini-environments. Geneva:
International Organization for Standardization.

ISPE (2009) Good practice guide: Heating, ventilation, and air

conditioning (HVAC). Bethesda, MD: International Society for
Pharmaceutical Engineering.

Naughton, P. (2016) Air change rates: Philosophy and practice.

Presented at the 2016 ASHRAE Annual Conference, St. Louis,
MO, June 25–29.
 Design Basics of Pharmaceutical Cleanrooms 115

Sun, W. (2020) Controlled environments-room pressure, airtight­

ness, flow offset and pressurization strategies. ASHRAE Journal
December.

Sun, W. (2018) Cleanroom airlock performance and beyond.

ASHRAE Journal February.

Sun, W. (2003) Development of pressurization airflow design criteria

for spaces under required pressure differentials. ASHRAE
Transactions Vol. 109.

Sun, W. et al. (2017) ASHRAE Design Guide for Cleanrooms:

Fundamentals, Systems, and Performance. Atlanta: ASHRAE.

ABOUT THE AUTHOR

Mr. Sun has been working in the US as an engineer for 28 years
in building mechanical systems design, HVAC, consulting, training
and research in his current position as the President at Engsysco, Inc.
Mr. Sun is a Fellow, distinguished lecturer, a member of the
nominating committee and chairman of the technology transfer
committee at the American Society of Heating, Refrigerating and Air
Conditioning Engineers (ASHRAE).

He was the chairman of TC 9.11 committee for clean spaces as

well as a member of TC 9.6 committee for healthcare facilities He
was also the President of the ASHRAE Detroit Chapter in 2010–2011.
As the principal author, his 450-page new technical book ASHRAE
Design Guide for Cleanrooms was published in 2017. He led over 20 co-
authors and spent six years in accomplishing this major achievement.

In other leadership roles, Mr. Sun served as the President of

Institute of Environmental Science and Technology (IEST) Society
in 2016–2017 in charge of all society leadership and management
116 Cleanroom Contamination Prevention and Control

responsibilities. He is also the USA Delegate for ISO cleanroom

standards 14644 on behalf of the USA since 2012. Mr. Sun is also the
chairman of the National Environmental Balancing Bureau (NEBB)
for the cleanroom performing testing standard (CPT).
 4

CLEANROOM DESIGN QUALITY

CONTROL AND DOCUMENTS
REVIEW

Wei Sun
Engsysco, Inc.
Farmington Hills, MI
USA

Pharmaceutical product manufacturing facilities require careful

assessment of many factors, including architectural, structural,
civil, heating, ventilation, and air conditioning (HVAC), electrical,
controls, process equipment, utility services and room operations.
Flows of equipment, personnel, material, product and waste must
also be considered along with system flexibility, redundancy,
maintenance and shutdown strategies. It is important to involve
design engineers, contractors, commissioning staff, and personnel
involved in quality control, validation, operations, maintenance, and
production representatives during the conceptual stage of design.

117
118 Cleanroom Contamination Prevention and Control

One factor that differentiates pharmaceutical processing suites

from other clean spaces is the requirement to meet government
regulations and inspection for product licensing; it is important
to include the appropriate regulatory considerations early in
the conceptual design phase. In the United States, regulatory
requirements and specification documents such as Food and Drug
Administration’s (FDA) current good manufacturing practice (cGMP)
for finished pharmaceuticals and for sterile products, and others
such as cleanroom standards and guidance including International
Society for Pharmaceutical Engineering (ISPE) guidelines describe
cGMP requirements. The goal of cGMP is to achieve a proper and
repeatable method of producing therapeutic, medical, and similar
products free from microbial and particle contaminants.

USER REQUIREMENTS SPECIFICATION

FOR DESIGN DOCUMENTS
Involved parties or regulated companies should have established
methods for developing and reviewing a formal User Requirements
Specification (URS) which should capture both the fundamental
aspects and scope of the users’ requirements. Users should be
required to review and approve these requirements that should
be objectively stated such that they can be verified during
design, testing and commissioning to assure design, installation,
operating, and performance quality. Alignment with objectives of
any company strategy or master plan should be confirmed. The
requirements should adequately focus on process and products in
addition to engineering aspects. A common URS may include, but is
not limited to the general scope, process description, methodology
of cleaning, sterilization, sanitization, design requirements of
architectural, mechanical, electrical, control and automation systems,
environmental, health and safety (EHS) impacts and control, quality
control, and revision history, among other variables. This chapter
will focus more on engineering design requirements.
 Cleanroom Design Quality Control and Documents Review 119

PROGRESSIVE FLOW OF DESIGN PHASES

Building design documents are often produced collectively by
architects and engineers (in structural, civil, mechanical, electrical,
process engineering disciplines, and other disciplines). For a typical
cleanroom building or a clean space design process, there are five
phases to architectural and engineering design services:

• Schematic design.
• Design development.
• Construction documents.
• Bidding.
• Construction administration.

In terms of the progressive percentage toward 100% completion for

final construction documents, Table 1 below gives a general outline
of the timelines, efforts and deliverable by a design firm, however
work may vary from project to project.

Phase 1 – Schematic Design

The owner’s project requirements are developed during planning
with the client to establish baseline criteria for facility function,
performance and maintainability. Schematic Design (SD) is the first
phase of actually designing the project, the Architect and Engineer
(otherwise called an A/E firm) discuss project requirements with the
client as provided by the client. Then A/E team establishes the size,
shape, and looks of the building, sub-spaces in all floors, down to
each functional room. SD phase has a great deal of brain storming,
sketching and modeling including meetings with the client to
outline the entire project. Once the basic design ideas and associated
approaches are presented with schematic drawings to the client, the
A/E and client will agree, in writing, to proceed to the next phase of
design. One of the important documents typically drafted by the A/E
firm for the client during this phase is called Basis of Design (BOD),
this document is an 8.5”x11” size Word/PDF document in the US.
120 Cleanroom Contamination Prevention and Control
Table 1 The five phases of architectural and engineering design

Progress in
Documents deliverable
Phase Effort percentage toward
by design firm
completion of design
Schematic Design (1) Basis of design (initial)
1 0–20%
(SD) (2) Drawings for progress review
Design
2 20–50% Drawings for progress review
Development (DD)
(1) Basis of design (updated)
Construction (2) Drawings issued for bidding
3 50–100%
Documents (CD) and construction
(3) Specification book
Q/A to bidders/contractors
4 Bidding (BD)
(optional)
(1) Review of shop drawing
Construction submittals
5
Administration (CA) (2) Punch list (before end of
construction)

The purpose of the BOD narrative is to document the reasoning

and decisions made during the schematic design phase of a
project. The BOD narrative is the design team’s implementation
of the owner project requirements. It presents the basic rationale
and assumptions, criteria, logic, and considerations developed in
evaluation of building A/E systems design. The BOD is created
during the schematic design phase and is continually updated during
the Design Development (DD) phase through the Construction
Document (CD) phase. However, for small projects, an owner may
choose to skip this document and ask a design team to proceed with
actual schematic drawings only.

Phase 2 – Design Development

In DD phase, the A/E firm revises, details and refines the initial
schematic drawings based on the client’s feedback and comments
from the SD phase, capturing more specifics and details. Detail
engineering designs will start on the established building structure
with early-stage HVAC, plumbing, fire protection, electrical, control,
 Cleanroom Design Quality Control and Documents Review 121

process and automation systems, energy analysis and any other

project-specific systems. At the end of DD, a good deal of product
selection and systems design should be progressing. Once the A/E
firm provides the client with the drawings, the architect and owner
will agree and move ahead to proceed to the next phase of design.

Phase 3 – Construction documents

With review and feedback from the client, the A/E firm prepares
more detailed drawings, notes, and most technical specifications
necessary for bidding, construction, and permit application. The CD
phase is the largest of all the phases for the A/E firm’s work efforts.
The A/E firm will finalize all the technical drawings and engineering
including detailing of the systems of HVAC, plumbing, electrical,
civil, utility, process, instrumentation, control, automation, and
energy, and other critical parameters. These will be prepared for
the documents that will be readied for used in pricing, biding and
construction, all products and materials are selected and scheduled
at this point. Once the construction documents are presented to the
client and mutually agreed upon, these documents are ready for
bidding and application for permit.

There are often three parts of Construction Documents:

• Most updated BOD.

• A set of drawings.
• Specifications.

Drawings (commonly in 42”×36” or 36”×24” sized sheets in the US)

are the most important deliverable CD which is a critical part of the
contract that graphically shows the scope, extent, and character of
the work to be performed by contractors. Specifications (typically a
thick book in 8.5”×11” size) are another part of the deliverable CD that
consists of written requirements for materials, equipment, systems,
standards, and workmanship as well as certain administrative
requirements and procedural matters applicable to the work.
122 Cleanroom Contamination Prevention and Control

Phase 4 – Bidding
Bidding (BD) is where the owner prepares to select the contractors
in various trades for the job and signs contracts to proceed with
construction. Multiple contractors may submit bids for the job or the
client can directly hire a contractor without getting competitive bids.
The A/E firm can review submitted bids and provide comparison
and analysis prior to submitting contractors to the client.

Phase 5 – Construction administration

The Construction Administration (CA) phase is the last phase on a
project. Even though this phase is the longest scheduled phase, it
is not the majority of the A/E firm’s work. The A/E firm typically
visits the job site periodically to witness the construction progress
and ensure the contractors are following the CDs. The A/E firm is
available to answer questions and provide additional information
to issues that may arise. Another service from the design firm is the
review of shop drawing submittals. During this phase it is common
that some additional A/E firm services may be solicited due to change
orders to address any unknown or unforeseen field conditions.

DESIGN QUALITY CONTROL PLAN

ON DESIGN DOCUMENTS
Quality control of design documents is extremely important, design
drawings and specifications may contain non-effective, deficient,
inferior performance or even malfunctioning concepts, erroneous
calculations, incorrect diagrams, details or schedules. These
unidentified issues will not be reverted or corrected automatically
later on, during testing, commissioning, validation, certification
or qualification phases. All architectural and engineering design
documents should be reviewed at proper design phases to ensure
that any risk, errors and omissions can be identified in a timely
manner, corrected and avoided before the final design drawings
are sealed for fabrication, installation and construction. As such, a
 Cleanroom Design Quality Control and Documents Review 123

detailed Project Quality Plan (PQP) on design documents should be

developed accordingly.

DESIGN REVIEWS
A formal review plan should be implemented for reviewing the
design against objectives and intentions to ensure that adequate
quality is delivered at optimized cost and energy consumption.
Designers should come to a consensus with users that a review plan
for quality control of drawings and specifications at proper stages
by independent experts is required.

Review expert selection

The design should be reviewed for each discipline at pre-defined
milestones or timelines, such as at a proper percentage of the design
progress by an independent third party. The third-party reviews
should be conducted independently of the original design teams
and companies so that the reviews can produce unbiased comments.

The third party of consultants or industry experts should be

highly recognized and adequately accomplished in the respective
industry for the subject matters to be able to identify, reveal or
disclose unforeseen errors, omissions and mistakes on the drawings
and documents which can be reported on in a timely manner and
be either clarified, addressed, updated, improved, and/or corrected
by the original design teams prior to progressing into later phases
where design changes may be difficult to address.

Review mechanism
Review category for each discipline should be identified and
established by the end users with industry experts in the design
quality plan. Review method should be defined according to type,
scale and risk by skilled experts to challenge the design in detail and
ensure that specified user requirements are delivered.
124 Cleanroom Contamination Prevention and Control

The methods should identify all relevant codes, regulations,

technical norms, and standards that may require compliance, and
formally confirm applicability and conformance. The review should
ensure that all factors are likely to be significant at a given review
stage.

For example, a review can identify if:

• The design meets the compliance aspects of local and interna-

tional codes, standards and industry regulatory requirements.

• The specified materials are the most applicable and suitable for
the intended proposes.

• Any hazard, fire and explosion risks exist, if any negative

impacts on environmental, health and safety aspects may occur.

• The airflow distribution, patterns and air change rate can achieve
an intended International Organization for Standardization
(ISO) class and GMP grade without over-supplying energy.

• Room pressures as indicated on drawings can actually be

achieved based on current HVAC configuration, P&ID diagrams,
and room air balances, etc.

Review outcome
Review should have appropriate method for recording and
disseminating the results of design review and managing any
consequent changes. Sequential review reports should confirm the
continuing suitability of the design.
 Cleanroom Design Quality Control and Documents Review 125

REFERENCES
ASHRAE (2019) ASHRAE Handbook – HVAC Applications. American
Society of Heating, Refrigerating and Air-Conditioning
Engineers, Inc. Atlanta, GA. Chapter 19, Clean Spaces.

FDA (2008) Current good manufacturing practice for finished

pharmaceuticals. 21 CFR 210, 211. Code of Federal Regulations,
U.S. Government Printing Office, Washington, D.C.

FDA (2004) Guidance for industry: Sterile drug products produced

by aseptic processing – Current good manufacturing practice.
U.S. Department of Health and Human Resources, Food and
Drug Administration, Washington, DC.

IEST-RP-CC012.4 (2020) Considerations in Cleanroom Design. Institute

of Environmental Sciences and Technology, Arlington Heights,
IL.

ISO (2016) ANSI/IEST/ISO Standard 14644-4:2001 (R2016).Clean-

rooms and associated controlled environments – Part 4: Design,
construction and start-up. International Organization for Stand-
ardization, Geneva, Switzerland.

Schneider, R.K. (1995) Practical Cleanroom Design. Business News

Publishing Company, Troy, MI.

Sun, W. et al. (2017) ASHRAE Design Guide for Cleanrooms. ASHRAE

Technical Book. ASHRAE, Atlanta, GA.

Whyte, W. (ed.) (1991) Cleanroom Design. John Wiley & Sons,

Chichester, England.
126 Cleanroom Contamination Prevention and Control

ABOUT THE AUTHOR

Mr. Sun has been working in the US as an engineer for 28 years
in building mechanical systems design, HVAC, consulting, training
and research in his current position as the President at Engsysco, Inc.
Mr. Sun is a Fellow, distinguished lecturer, a member of the
nominating committee and chairman of the technology transfer
committee at the American Society of Heating, Refrigerating and Air
Conditioning Engineers (ASHRAE).

He was the chairman of TC 9.11 committee for clean spaces as

well as a member of TC 9.6 committee for healthcare facilities He
was also the President of the ASHRAE Detroit Chapter in 2010–2011.
As the principal author, his 450-page new technical book ASHRAE
Design Guide for Cleanrooms was published in 2017. He led over 20 co-
authors and spent six years in accomplishing this major achievement.

In other leadership roles, Mr. Sun served as the President of

Institute of Environmental Science and Technology (IEST) Society
in 2016–2017 in charge of all society leadership and management
responsibilities. He is also the USA Delegate for ISO cleanroom
standards 14644 on behalf of the USA since 2012. Mr. Sun is also the
chairman of the National Environmental Balancing Bureau (NEBB)
for the cleanroom performing testing standard (CPT).
 5

CLEANROOM AIRFLOW
VALIDATION IN DESIGN PHASE

Wei Sun
Engsysco, Inc.
Farmington Hills, MI
USA

Airflow inside a cleanroom is the primary carrier of heat, moisture,

and contaminants such as viable and nonviable particles. The
distribution of supply air predominantly determines the result­
ing air velocities, temperatures, and concentration of particles at
various locations in a cleanroom, and such distribution in turn
determines thermal comfort and air cleanliness class. Removal of
viable and nonviable particles from a cleanroom is mainly achieved
through return and/or exhaust air outlets. Satisfactory thermal
comfort for occupants, energy saving, and maintaining the desired
cleanliness are mutually competing goals. Obtaining these goals by
optimizing various design and operating parameters of cleanroom
air distribution systems is not an easy task especially during the
design phase.

127
128 Cleanroom Contamination Preventiion and Control

FACTORS IMPACTING CLEANROOM

AIRFLOW PATTERN AND RESULTING
ISO CLASS AIR CLEANLINESS
Airflow patterns, temperature, and particle distribution in a
cleanroom depend on several interconnected factors, including:
• Location of supply diffusers.
• Supply air flow rates (air change rates) and associated diffuser
throws.
• Supply air temperature, size and locations of room return and
exhaust.
• Leakage areas and associated airflow rates.
• Locations.
• Location and size of obstructions to airflow.
• Relative location and strength of particle-generating entities in a
cleanroom.

HOW TO ASSURE REQUIRED OUTCOMES

DURING DESIGN PHASE
The design phase of a new pharmaceutical cleanroom suite is
typically unique by either its location, physical size, shape, process,
or intended ISO class, etc. Prior success of a cleanroom design
concept cannot guarantee that a new design will produce a similar
successful outcome where the location, physical size, shape, process,
heating, ventilation, and air conditioning (HVAC) system, particle
emission, etc. could be totally different.

Smoke visualization is a great tool to illustrate the possible

outcomes of airflow patterns and distribution after a cleanroom is
fully constructed, which is required by ISO cleanroom Standard
14644 Part 3. Smoke studies/airflow visualization is recommended
under static and dynamic conditions per the FDA in Grade A,
 Cleanroom Airflow Validation in Design Phase 129

devices and critical processing rooms. This includes addressing

aseptic interventions and requires a written assessment of airflow
patterns; video documentation has been found to be a useful aid
in this regard. However during design phase when the future
cleanroom is still on paper on a drawing board, nothing can be
physically tested. Therefore it is important to acquire other means for
airflow verification before the design paper documents are finalized
for the construction phase. Use of un-validated design documents
directly for construction could risk costly mistakes under regulatory
inspections for airflow pattern effectiveness.

The pre-construction simulations and animations on airflow and

particle movement analysis can help avoid unforeseen design errors,
deficiencies and ineffectiveness before obtaining the optimized
design approaches/documents to be used for construction. In this
situation, analysis of various realistic scenarios through comput­
ational fluid dynamics (CFD) simulations is an effective alternative.

AIRFLOW VERIFICATION REQUIREMENTS

BY ISO STANDARD AND DESIGN GUIDELINES
The newly published ISO Cleanroom Standard 14644 “Cleanrooms
and associated controlled environments – Part 4. Design, construc-
tion and start-up” (ISO, 2001) indicates the importance of having
airflow validation during design phase in its Annex B as below:

Computational fluid dynamics (CFD) can be applied to optimize

designs by assessing the expected performance of the airflow in a
cleanroom or clean air device design. This modelling tool can provide
information for a designer to adjust the design to achieve more effective
and efficient results.

The ASHRAE Applications Handbook (ASHRAE, 2019) indicates that

below regarding CFD simulation:

CFD analysis can predict airflow patterns, resulting temperature

distribution, particle concentration, relative humidity distribution,
130 Cleanroom Contamination Preventiion and Control
and resulting thermal comfort of occupants in confined spaces such
as cleanrooms. In cleanroom design analysis, it is used to predict the
effects of room pressurization (i.e., relative supply and return airflow
rates, locations of supply and returns, particle generation rate on
the distribution of cleanliness in a room). CFD analysis can help provide
deep insight into real-life operation of cleanroom at the conceptual
design stage, which in turn can help in optimizing the operating para­
meters and in reducing the first and operating costs of HVAC systems.

The Institute of Environmental Sciences and Technology (IEST) in

its Recommended Practices of RP-CC012 (IEST, 2020) under the title of
“Considerations in Cleanroom Design” indicates that:

CFD can be utilized for both new construction and upgrades to

existing facilities. In the case of new construction, one can utilize 3D
of the room or space and the mechanical parameters should be part
of the design documents. 3D models of any equipment and furniture
are also needed. This allows for a full 3D representation of the space,
along with air supply, air return, exhaust data. The air flow patterns
through the room and around the equipment can then be observed via
the model prior to construction. This provides a confirmation of the
design properly fitting the requirements saving dollars and time if the
design needs to be tweaked.

There are many guidelines and CFD application cases that are
available at various outline sources. CFD has become a key tool for
airflow visualization and validation during cleanroom design phase.

Though CFD is a very useful tool during design phase, it cannot

be substituted for in-situ air pattern analysis which is required by
the regulators.

In-situ air pattern analysis should be conducted at the critical

area to demonstrate unidirectional airflow and sweeping action
over and away from the product under dynamic conditions. The
studies should be well documented with written conclusions and
include evaluation of the impact of aseptic manipulations (e.g.,
in­terventions) and equipment design. Video documentation or
other recording mechanisms have been found to be useful aides
 Cleanroom Airflow Validation in Design Phase 131

in assessing airflow initially as well as facilitating evaluation of

subsequent equipment configuration changes. It is important to note
that even successfully qualified systems can be compromised by
poor operational, main­tenance, or personnel practices (FDA, 2004).

CFD PRINCIPLE
Computational fluid dynamics involves solution and analysis of
transport equations of fluid flow, heat transfer, and mass transfer
in indoor environments. The transport of continuity, mass,
momentum, energy, and contaminants’ species are governed by a
generalized conservation principle that can be described in the form
of a general differential equation. During this CFD procedure, first
the calculation domain (extent of space) is divided into a number of
discrete cells (non-overlapping control volumes) for the meshing,
such that there is one control volume surrounding each grid point.
Then, each governing differential equation is iteratively balanced
over each control volume to conserve the mass, momentum, energy,
and other physical entities. During the iterative process, the residual
error for each governing equation is monitored and reduced. This
process continues until the overall balance in the conservation of
all the governing entities is achieved up to an acceptable desired
level. Finally, such converged numerical solutions reveal a
detailed distribution of pressure, velocities, turbulence parameters,
temperature, concentrations of chemical, particle and microbial
(contaminants) species.

CFD PRACTICES FOR CLEANROOMS

CFD can be applied to optimize designs by assessing the potential
performance of a cleanroom design. This method can allow a
designer to adjust design parameters for better and more efficient
results, and predict the behavior of their systems for virtually any
scenario, and void some of the hidden pitfalls. In outline, as stated
in ISO 14644-Part 4 (ISO, 2001), the key points of this method are
as follows:
132 Cleanroom Contamination Preventiion and Control

• CFD requires, as an input, the information on geometry of the

cleanroom, equipment and process locations, personnel number
and gowning level, contaminants’ emissions and locations,
positions of air inlets and returns, exhaust and other boundary
conditions, and their airflow rates and air properties such
as temperature and humidity, etc. 3D CAD files are directly
imported to CFD to define the geometry of the objects being
studied.

• CFD allows visualization of airflow patterns and contaminant

concentrations within the space; enabling identification of areas
or spots that indicate poor performance, such as phenomena
of low velocity, eddy flow, high particulate concentration,
reversed flow of contaminated air that migrates into cleanroom
from corridor during door operation.

• CFD permits the modeling of spot locations of contaminated

air dispersed or migrated from contamination sources under
various HVAC control scenarios.

• CFD output allows calculation under various HVAC configur­

ations and airflow patterns to assess the operation of the
cleanroom at areas of concern, and to optimize the airflow
pattern and airflow rate effectiveness to achieve predicable
performances for the design.

The goal of CFD modeling and simulation is often to help end-users’

Design Qualification (DQ) process on the HVAC design, especially
for the airborne contamination control concept, CFD is utilized to
check if the design intention can meet the ISO classes, pharmaceutical
grades, airflow patterns and other intended objectives prior to
cleanroom construction and installations.
 Cleanroom Airflow Validation in Design Phase 133

APPLICATIONS OF CFD SIMULATION

AND ANALYSIS FOR CLEANROOMS
As indicated earlier, CFD can be applied to optimize designs by
assessing the expected performance of the airflow in a cleanroom
or clean air device design such as laminar air flow (LAF), isolator,
Restricted Access Barrier Systems (RABS), mini-environments, etc.
This modeling tool can provide information for a designer to adjust
the design to achieve more effective and efficient results. ISO 14644-
Part 4 (ISO, 2001) lists some important features of this technique as
follows:

• Conversion of the source strength and particulate concentration

limits from particles/unit time into mass concentrations/unit
time allows most CFD software to track particulate emissions
through a space.

• Enables the modeling of point location of contamination sources

at “worst case” locations and shows the impact of these sources
on critical locations.

• Allows visualization of airflow patterns and contaminant

concentrations within the space; enabling identification of areas
that have poor cleanroom performance (e.g., low velocity, high
particulate concentration, reflux (airflow pattern from less clean
to cleaner classified area), etc.).

• The output allows calculation of ventilation effectiveness at

critical locations within the space, to assess the operation of
the cleanroom at areas of greatest concern. The ventilation
effectiveness thus determined may be used as input to dilution
calculations.

• In order to be useful CFD requires, as an input, the

information on geometry, air volume flow rates, air inlets and
returns, exhaust and other boundary conditions. Effective
CFD modeling requires input of important data including
134 Cleanroom Contamination Preventiion and Control

information on room or zone geometry, air volume flow rates,

air inlet and return locations, aerodynamic performance of
supply air diffusers, exhaust and other boundary conditions.

In recent years, use of CFD for cleanroom airflow, cleanliness class

and room pressure predictions has become a common practice in
verification of cleanroom design concepts. Some verifications are
listed below, but are not limited to those that can be achieved:

• Simulate gaseous, microbial and particulate airborne contam­

inants in cleanrooms.
• Transient simulations to calculate purging time or recovery time
in worst case scenarios.
• Analysis of cleanroom performance at as-built, at rest and
operational states, and identify the recovery time after a
challenge occurs.
• Simulate adsorption suite and fermentation room.
• Predict particulate loading on exposed surfaces and airborne
concentration.
• Simulate various chemical, particle and/or microbial dispersion
locations, strength, patterns and controlled timing.
• Identification of recirculation zones and design features that
may lead to detrimental air flow.
• Airflow optimization for large raised-floor flat panel display
facility.
• Internal airflow optimization between enclosure and wafer
cassette mini-environment.
• ISO Class 1 cleanroom and indoor airborne molecular
contamination (AMC) control.
• Ultra-negative biosafety – 3/4 laboratories and control of air­
borne decontamination.
 Cleanroom Airflow Validation in Design Phase 135

• Simulation of chemical fume removal and gas leakage detection

and control in semiconductor facility.
• Grade A, B or C aseptic cleanrooms, in various suite configura-
tions.
• Passive or active RABS and their impacts to room airflow
patterns.
• RABS airflow interaction between inside chambers and back­
ground room during human intervention (door is opened).
• Airflow patterns and effectiveness inside isolator chambers with
vial conveyor operation.
• LAF and its impact to room airflow pattern due to side intake.
• Laminar flow hoods/clean benches.
• Various airlocks (cascading, sink, bubble, dual-compartment),
interlock and their transient performance during door operations.
• USP 797 suite for pharmaceutical compounding-sterile prepara-
tions.
• USP 800 suite for hazardous drugs-handling in healthcare
settings.
• Operating room airflow simulation and optimization.
• Isolation room airflow simulation and optimization.
• Lyophilizers and lyophilization areas.
• Vial filling areas.
• Particle migration through door operation due to transient room
pressure loss.
• Robotics.
• Cappers.
• Accumulators.
• Flash freezers.
136 Cleanroom Contamination Preventiion and Control

EXAMPLES OF CFD MODELING,

SIMULATION AND ANALYSIS
Geometry 3D modeling of cleanroom suite
(functional group of multiple cleanrooms)
Figure 1 shows a three-room cleanroom suite that accommodates an
ISO 7 gown room/airlock (equipped with demarcation bench, sink,
and rack), an ISO 6 prep room (with fume hood, biosafety cabinet,
perforated table), and an ISO 3 cleanroom (with perforated tables).
All rooms use either high-efficiency particulate air (HEPA) filters or
fan-filter units to achieve the required ISO classes by predicting the
proper levels of air change rates (ACRs) based on particle generation
information, room geometry, HVAC system confirmation, HEPA
supply air/return air placement, air change rate, etc. It is more
meaningful to build multiple rooms in a single CFD modeling so
that air migrations between rooms during door operation can be
investigated.

Figure 1 Geometry of a three-room cleanroom suite

Source: Author
 Cleanroom Airflow Validation in Design Phase 137

Analysis of room airflow patterns and effectiveness

Figure 2 shows a pharma filling room modeling. An isolator was
placed in the middle of the room, HEPA filters were placed on ceiling,
return air points were located at room corners with open-end return
ducts. However in this initial design concept, HEPA supply air may
hit down the floor and bounce back, so that upward eddy airflow
patterns were observed along the side-walls. This phenomenon
indicated that instead of multiple small return air points, a long
open-end return wall at each perimeter wall would be needed to
reduce or eliminate upward flows and let particles exit through low
walls more naturally. A re-configuration of HEPA filters and return
passages can be conducted to find out the optimized outcomes after
a few trials.

Figure 2 Pharma filling room modeling and airflow simulation

focusing on effectiveness of flow patterns and particle removal
passages in room

Source: Author
138 Cleanroom Contamination Preventiion and Control

Isolator’s LAF unit and vial mousehole exit

Figure 3 was intended to identify and illustrate the airflow interaction
between an LAF air stream and a mousehole air jet from an isolator’s
outfeed tray. Most LAF units use fan-filter unit to provide a vertical
velocity around 90 FPM (0.45 m/s) downward to blow off particles
that are emitted from pharmaceutical packaging line and conveyor
transportation of vials, bottles, syringes, cartridges or ampoules.
Besides, LAF units are often used in vial’s infeed and outfeed
sections for pre-cleaning and particle blow-off propose. Figure 3
shows that mousehole exit air jet was too strong and that the vertical
LAF airflow could be undesirably off sideway so that the clean air
cannot fully wipe out particles around the tray’s surface, therefore a
better mechanism needs to be found, and the air patterns should be
optimized through CFD trials in multiple scenarios.

Figure 3 Interaction between LAF vertical air stream and mousehole

horizontal air stream

Source: Author
 Cleanroom Airflow Validation in Design Phase 139

Analysis of particle migration through door opening

Figure 4 shows that a typical cleanroom door operation (opening
and closing) can trigger a transient pressure differential’s disruption
that causes undesirable particle migration direction across door
opening. Due to the loss of initial pressure differential ∆P, particles
could migrate from a higher concentration (dirtier) room to a lower
concentration (cleaner) room based on the mass diffusion principle.
This phenomenon is often in contradiction to the initial desire that a
higher pressure (cleaner) room can prevent particles migrating from
the lower pressure (less clean) room. For a specific cleanroom, CFD
trials in multiple scenarios can deliver an optimized outcome for
“contamination control during door operation”.

Figure 4 Air velocity contour during door operation: cause transient

pressure differential’s disruption and undesired particle migration
across door opening

Source: Author
140 Cleanroom Contamination Preventiion and Control

Analysis of airflow profiles in sectional

plane views of a filling room
Figures 5(A) and 5(B) illustrate some of vertical sectional views of
airflow velocity around an isolator. In these figures, it was found that
some recirculated flows occur along walls and eddy flows around
personnel, it seemed that the ceiling HEPA filters are not the only
dominant factor. Re-arrangement of HEPA filters and reduction of
personnel present may be necessary. Besides, additional horizontal
section plane cuts can be also developed to visualize air velocity
distribution at different room heights.

Figure 5(A) Vertical plane view Figure 5(B) – Vertical plane view
(east–west section) (north–south section)

Source: Author

Analysis of room particle concentration

at various room heights
Figure 6 below shows the non-variable particle (0.5 µm) distribution
in a horizontal section view at 3 feet height. Besides, the average
particle concentration (in particle counts per room volume) is
calculated automatically at various room heights. These efforts can
allow the design engineer and facility manager to estimate what
could be the resulting ISO air cleanliness class for optimization
 Cleanroom Airflow Validation in Design Phase 141

before the final design concept is to be set in concrete on drawings

for construction. One key benefit of CFD simulations is to review the
outcomes of many scenarios of possible design concepts, and none
of these initial concepts need to be built physically. Among these
scenarios, the design or facility engineer is able to pick the optimized
concept in terms of performance, cost, energy efficiency, etc.

Figure 6 Showing room particle concentration

at various room heights

Source: Author

Analysis of particle flow paths from ceiling

HEPA filters to sidewall return ducts
Figure 7 demonstrates that particles emitted from multiple personnel
can travel following the airflow paths from ceiling HEPA filters to
sidewall return ducts, however due to one side of the room has
more personnel than the opposite side, naturally more particles are
emitted on that side. Some return ducts take out more particles than
those from the rest return ducts. Meanwhile particles may not exit
smoothly, some eddy and recirculation flows are observed. These
eddy flows can be minimized by an increase of return points/ducts
to allow particle to exit as smooth as possible.
142 Cleanroom Contamination Preventiion and Control
Figure 7 Particle flow paths from ceiling HEPA filters
to sidewall return ducts showing recirculation of
particles prior to return points

Source: Author

REFERENCES
American Society of Heating, Refrigerating and Air-Conditioning
Engineers (ASHRAE) (2019) ASHRAE Handbook – HVAC
Applications. Chapter 19. Clean Spaces. Atlanta, GA: ASHRAE.

FDA (2008) Current good manufacturing practice for finished

pharmaceuticals. 21 CFR 210, 211. Code of Federal Regulations,
US Government Printing Office, Washington, DC.

FDA (2004) Guidance for industry: Sterile drug products produced

by aseptic processing – Current good manufacturing practice.
US Department of Health and Human Resources, Food and
Drug Administration, Washington, DC.

Institute of Environmental Sciences and Technology (IEST) (2020)

IEST-RP-CC012.4. Considerations in Cleanroom Design. Arlington
Heights, IL: Institute of Environmental Sciences and Technology.

ISO (2001) ISO 14644-4:2001 Cleanrooms and associated controlled

environments — Part 4: Design, construction and start-up
 Cleanroom Airflow Validation in Design Phase 143

International Organization for Standardization, Geneva,

Switzerland.

Schneider, R.K. (1995) Practical Cleanroom Design. Troy, MI: Business

News Publishing Company.

Sun, W. et al. (2017) ASHRAE Design Guide for Cleanrooms. ASHRAE

Technical Book. Atlanta, GA: ASHRAE.

Whyte, W. (Ed.) (1991) Cleanroom Design. Chichester, England: John

Wiley & Sons.

ABOUT THE AUTHOR

Mr. Sun has been working in the US as an engineer for 28 years
in building mechanical systems design, HVAC, consulting, training
and research in his current position as the President at Engsysco, Inc.
Mr. Sun is a Fellow, distinguished lecturer, a member of the
nominating committee and chairman of the technology transfer
committee at the American Society of Heating, Refrigerating and Air
Conditioning Engineers (ASHRAE).

He was the chairman of TC 9.11 committee for clean spaces as

well as a member of TC 9.6 committee for healthcare facilities He
was also the President of the ASHRAE Detroit Chapter in 2010–2011.
As the principal author, his 450-page new technical book ASHRAE
Design Guide for Cleanrooms was published in 2017. He led over 20 co-
authors and spent six years in accomplishing this major achievement.

In other leadership roles, Mr. Sun served as the President of

Institute of Environmental Science and Technology (IEST) Society
in 2016–2017 in charge of all society leadership and management
responsibilities. He is also the USA Delegate for ISO cleanroom
standards 14644 on behalf of the USA since 2012. Mr. Sun is also the
chairman of the National Environmental Balancing Bureau (NEBB)
for the cleanroom performing testing standard (CPT).
 6

AIR FLOW VISUALIZATION:

THE MOST MISUNDERSTOOD AND
UNDERUTILIZED CONTAMINATION
CONTROL TOOL

Morgan Polen
Microrite, Inc.
San Jose, CA
USA

AIR FLOW VISUALIZATION FOR PHARMACEUTICAL

AND OTHER MEDICAL PRODUCT CLEANROOMS
Air flow visualization (air pattern analysis) is a science, not unique
to pharmaceutical cleanrooms or the medical product industry.
Air flow visualization is used in many different industries, such as
aerospace, automotive, electronics manufacturing and data centers.

Understanding the effects of air flow over objects such as airplane

wings or automobiles is critical for design and performance review. In
these industries it is extremely important that the visualization cloud
(made up of tracer particles), faithfully follow the streamlines of the
air flow, allowing the analysis and evaluation of the physical (actual)
air flow patterns against design and operational requirements.

145
146 Cleanroom Contamination Prevention and Control

Various industries utilize cleanrooms to control contamination.

Cleanrooms are high technology environments that require
appropriately pre-conditioned high efficiency particulate air (HEPA)
filtered air, supplied in a consistent manner with a sufficient volume
and in a direction that provides a contamination control effect.

Air flow visualization studies (aka smoke studies/air pattern

analysis) were one of the original tools used in the testing and
qualification of clean air devices and cleanrooms since the 1940s.
Cleanroom technology has evolved from open cleanrooms in the
1970s to the use of isolation technology for critical manufacturing
and is commonly used today. Air flow visualization technology has
also evolved from the use of cigarettes capturing “laminar” air flow
patterns on polaroid cameras in the early days, to more modern “in-
situ air pattern analysis” capturing “unidirectional” air flow patterns
on high definition digital recording media.

Pharmaceutical and medical product manufacturing cleanrooms

require greater microbial and particulate control than cleanrooms
used in other industries. A key aspect of this environmental control
is the contamination control effect of HEPA/ULPA (ultra low
particulate air) filtered air moving in suitable volume and direction
to prevent the settling of contamination on products, product
handling surfaces and all other surfaces in the cleanroom.

In non-unidirectional flow cleanrooms, the contamination

control effect is achieved by the over pressurization of the cleanroom
by continually moving clean (HEPA/ULPA filtered) air through the
cleanroom and out via strategically placed exhausts or air returns.
This effect prevents an ingress of contamination from external
sources, while sweeping contamination out of the cleanroom via
suitably placed exhausts or air returns in order to maximize the
contamination control effect of clean air.

In areas where lower contamination levels are required (≤ISO

Class 6) the contamination control effect is enhanced by using
unidirectional air flow. This is where air flow is directed in a single
 Air Flow Visualization 147

direction providing a sweeping action over surfaces, displacing any

contamination intrinsic to the process, out of the critical area, while
preventing contamination from outside the process from entering
the critical area.

Many pharmaceutical facilities utilize combination flow

conditions, where a unidirectional flow zone interfaces with a non-
unidirectional flow zone. A common example would be a Grade A
filling zone inside a restricted access barrier systems (RABS)
enclosure surrounded by a Grade B background cleanroom. Air flow
visualization of the interface between these two zones is exceptionally
important (and a regulatory expectation) as it provides a physical
evaluation of the contamination control effectiveness of the RABS
enclosure, the cleanroom and the integration of these two systems.

WHAT IS AIR FLOW VISUALIZATION

(AKA SMOKE STUDIES)?
Air flow visualization is the science of making air flow patterns visible.
Because air is transparent, air flow patterns are invisible to the naked
eye. Many high technology industries such as electronics, chemical,
automobile, aerospace and defense, use air flow visualization as an
engineering tool to optimize design and performance of mechanical
systems. It is important to understand how various industries perform
air flow visualization in order to develop the best possible methods
to characterize air flow patterns in pharmaceutical cleanrooms.

The most common method to make air patterns visible is the

tracer particle method. The tracer particle method utilizes the addit-
ion of millions of tracer particles into the air stream. The resulting
cloud of tracer particles makes the air patterns visible. It is important
that the tracer particles faithfully follow the streamlines of the air
flow, allowing the analysis and evaluation of the physical (actual)
air flow patterns against design and operational requirements.
(The term “smoke study” is often used synonymously for air
flow visualization as initially these studies utilized smoke sticks,
cigarettes, etc.)
148 Cleanroom Contamination Prevention and Control

In cleanrooms air flow patterns play an important part of

establishing a “contamination control effect”. Air flow visualization
provides a tool that allows for the evaluation of air patterns in
both unidirectional and non-unidirectional flow cleanrooms. For
standardization purposes the ISO 14644 (ISO, 2005; 2015a, b) series
of International Cleanroom Standards lists “airflow direction test
and visualization” as an optional test for general (non-medical
product) cleanrooms and provides some guidance on methods and
equipment specifications. It is important to note, in medical product
cleanrooms, that the scrutiny of air flow patterns in aseptic operations
is a regulatory requirement. The Food and Drug Administration
(FDA), United States Pharmacopeia (USP), Pharmaceutical
Inspection Convention and Pharmaceutical Inspection Co-operation
Scheme (PIC/S), European Medicines Agency (EMA) and World
Health Organization (WHO) require air flow visualization studies
for aseptic areas, with written conclusions and suggest including
video documentation.

AIR FLOW VISUALIZATION IS A TOOL

FOR CONTAMINATION CONTROL
It is important that in medical product cleanrooms, dislodged
particles should not be redeposited on products, personnel or work
surfaces. Because human beings are a constant source of particulate
and microbial contamination, a pragmatic and scientific approach
must be deployed to prevent contaminated medical products. Air
flow visualization as applied to the science of contamination control
provides valuable information in terms of assessing contamination
risk. Understanding air patterns in all cleanrooms inclusive of
equipment and personnel movements is a fundamental component
of holistic contamination control.

The necessity of having a broad range of controls such as

cleanrooms, barrier systems, sterilization, sterile gowning, aseptic
practices, and risk based environmental monitoring is due to the
seriousness contamination poses to patients. It is important to note
the impossibility of maintaining a sterile cleanroom environment that
 Air Flow Visualization 149

has humans working in it for any period of time. Even with the most
technologically updated facility designs and rigid sterile cleanroom
gowning practices, human borne, and environmental contamination
is a constant threat to aseptically produced products. It is difficult to
fully eliminate microbial and particulate contamination in the entire
operating cleanroom, however, through the use of all these controls
and the use of robust, appropriately clean air flow, contamination
can be prevented from reaching products and product contact
surfaces for the duration of aseptic operations.

Because no amount of testing and monitoring can guarantee

a contamination free aseptic environment, current good manu­
facturing practice (cGMP) states that to minimize the risk of contam­
ination, air flow visualization should be utilized to optimize and
qualify the contamination control effect of the air flow under all
operating conditions.

The actual (or physical) contamination control effect of

cleanroom air flow can only be properly understood when it is
visually represented. From this visual representation and analysis:

• Engineering can optimize cleanroom\barrier system design and

integration.

• Manufacturing can optimize operator’s positions, movements

and sequences to reduce the risk of operator generated
contamination near product or product contact surfaces.

• Quality control and validation can review changes and create

a suitable means of documenting static and dynamic in situ air
pattern analyses once the optimization of the cleanroom/barrier
systems and the operator’s movements has been defined.

As regulatory bodies are expected to review air flow visualization

videos, it is important to work out any shortcomings in the system
before attempting the documented in situ air pattern analysis.
150 Cleanroom Contamination Prevention and Control
Figure 1 Image from a “smoke study”

“It is useful to assume that the operator

is always contaminated while operating
in the aseptic area.
If the procedures are viewed from
this perspective, those practices which are
exposing the product to contamination
are more easily identified.”
Hank Avallone, 1988

“A gowned operator may release as many as

10,000 CFU/hour or more…
(Reinmuller et al., 1996)

Note: Smoke is moving over the operator’s head and hands in the direction of the filling
needle holder and the turntable. This filling line had failed several media fills.

Source: Ljungqvist and Reinmüller (1996)

Too often these studies are approached only as a regulatory

requirement and not as a contamination control tool which can help
reduce the risk of contamination to products and product contact
surfaces. Air flow visualization studies are an extremely important
tool in assessing the physical contamination control effect of air
patterns and should be used to identify risk based environmental
sampling locations.

Based upon the author’s experience, FDA 483 observations

and warning letters, the performance of these studies is not well
understood.

TYPES OF AIR FLOW IN CLEANROOMS

A cleanroom’s contamination control effect is achieved by the use of
appropriately conditioned and filtered air delivered via one of the
following air flow concepts:
 Air Flow Visualization 151

• Non-unidirectional air flow

(See Figure 2.) For non-unidirectional flow cleanrooms or clean
zones the contamination control effect is achieved by the over
pressurization and dilution of the cleanroom by continually
moving clean (HEPA/ULPA filtered) air through the cleanroom/
clean zone and out via strategically placed exhausts or air
returns. This effect prevents an ingress of contamination from
external sources, while sweeping contamination out of the
cleanroom via suitably placed exhausts or air returns in order to
maximize the contamination control effect of clean air. This type
of cleanroom air flow is typical of cleanrooms with ISO Class 7,
8 or 9 classification.

ISO-14644-1:2015 (ISO, 2015a)

“Non-unidirectional Air Flow: Air distribution where the supply
air entering the clean zone mixes with the internal air by means of
induction.”

Air flow visualization of non-unidirectional air flow cleanrooms

and clean air devices is useful in characterizing the mixing of
clean air by providing a visual representation of the dilution
effect and the ability of the air returns or exhaust to control
contamination rates.

Visual representation of air patterns in non-unidirectional air

flow cleanrooms can identify undesirable areas of excessive
turbulence, eddy currents near critical operations.

It is exceptionally important to evaluate non-unidirectional

air flow cleanrooms for any deviant air patterns created by
equipment, doors or passageways. Cooling fans on computers,
human machine interfaces (HMIs), and other equipment must
be evaluated from an air flow pattern disturbance as well as a
possible source of contamination. For medical products the
author recommends air flow visualization studies be carried out
for all cleanrooms as part of the qualification of the cleanrooms.
These studies should be used in risk assessment, environmental
monitoring plans and the overall contamination control strategy.
152 Cleanroom Contamination Prevention and Control

• Unidirectional air flow

(See Figure 3.) For unidirectional flow cleanrooms or clean zones,
the contamination control effect is achieved by displacement
with clean air. Where the air flow is directed in a single direction
providing a sweeping action over surfaces, displacing any
contamination intrinsic to the process, out of the critical area,
while preventing contamination from outside the process from
entering the critical area.

ISO-14644-1:2015 (ISO, 2015a)

“Unidirectional airflow: Controlled airflow through the entire cross-
section of a cleanroom or a clean zone with a steady velocity and
airstreams that are considered to be parallel.”

Note that this type of airflow results in a directed transport of

particles from the clean zone.

FDA Sterile Aseptic Guide 2004 (FDA, 2004)

“Unidirectional flow – An airflow moving in a single direction, in a
robust and uniform manner, and at sufficient speed to reproducibly
sweep particles away from the critical processing or testing area.”

Air flow visualization of unidirectional air flow cleanrooms

and clean air devices is an inspectorate expectation but should
be considered as an engineering tool and part of the validation
(Factory Acceptance Test (FAT), Site Acceptance Test (SAT)) of
RABS, isolators and addressed in User Requirements Specific­
ations (URS). These studies should be used in risk assessment,
environmental monitoring plans and the overall contamination
control strategy.

• Combination air flow

(See Figure 4.) Many pharmaceutical and medical product
cleanrooms use a combination of air flows, utilizing non-
unidirectional air flow in less critical locations and relying on
unidirectional flow in critical areas for sterile or aseptic processing.
 Air Flow Visualization 153
Figure 2 Non-unidirectional Figure 3 Unidirectional flow Figure 4 Combination
flow cleanroom concept cleanroom concept flow cleanroom concept

Drawings generated by Microrite

Airflow patterns for cleanrooms of ISO Class 5 and cleaner in

operation are often unidirectional, non-unidirectional is typical
for cleanrooms of ISO Class 6 and less clean in operation. For all
airflow concepts removal of contamination as close as possible
to the source is important.

The EU or PIC/S document “GMP Annex 1 Manufacture of Sterile

Medicinal Products” (EU GMP, 2008) creates a grade system
for cleanrooms, that assigns additional criteria to cleanrooms
above and beyond the ISO 14644 standards. The grade system
establishes occupancy states, gowning expectations by grade,
viable and non-viable particle limits based upon grade and
occupancy state (either at rest or operational) as well as the
types of operations typically associated with each grade. Grade
A is expected to be unidirectional and Grade B may be either
unidirectional or non-unidirectional, depending upon its
proximity to the critical area.

As it is a regulatory expectation, air flow visualization studies

are used to evaluate the interface between Grade A (ISO Class 5)
environments and Grade B (ISO Class 7) environments in order
to demonstrate the maintenance of unidirectional airflow across
the whole of the Grade A area.
154 Cleanroom Contamination Prevention and Control

First air concept

The concept of “first air” is extremely important in the evaluation of
air flow visualization studies. First air describes the air exiting the
HEPA filter or HEPA diffuser in a unidirectional air stream that flows
directly over product or product contact surfaces prior to contacting
anything else. The “first air” concept is critical to aseptic and sterile
operations. “First air” specifically refers to what the air touches
“first”. There are no objects or persons in the air flow path between
the HEPA filter diffuser and the product or product contact surfaces,
including equipment frames, sensors, light fixtures. Unidirectional
air flow that contacts equipment or operating personnel is no longer
“first air” and should be noted in the air pattern analysis.

Figure 5 Illustration of the “first air” concept,

when the RABS doors are closed

Drawings generated by Microrite

• Non-desirable air flow patterns

Any discussion on air flow visualization should involve the
inclusion of non-desirable air patterns. Deviant air patterns are air
patterns that have a detrimental effect on contamination control.
 Air Flow Visualization 155

In unidirectional flow cleanrooms, eddy currents, vortexes and

excessive turbulence would be considered deviant air patterns
that should be avoided.

While non-unidirectional flow cleanrooms require the mixing

of clean air with room air, turbulence and some eddy currents
are desirable to control contamination rates in order to get good
mixing of clean air and to prevent the settling of particles on
surfaces. In unidirectional flow cleanrooms, deviant air may be
air patterns that move from a contaminated location to a more
critical location, such as upward moving air flow in the occupied
space of a Grade A/B aseptic filling line.

Ceiling mounted air returns

Figure 6 Impact of ceiling mounted air returns in cleanrooms

Large particles (>5.0 µM) are too heavy to be transported up to the ceiling.
Ceiling mounted returns keep larger particles in the cleanroom air longer, increasing the
risk of deposition onto operator’s clothing.
Drawing generated by Microrite

As the purpose of air returns in cleanrooms is to remove dirty

contaminated air, placing an air return in the celling is detrimental
to the contamination control effect (see Figure 6). As larger particle
contamination (particles greater than 5 micron in size) are affected
156 Cleanroom Contamination Prevention and Control

by gravity, these particles cannot be effectively removed via ceiling

mounted air returns.

Cleanroom short circuit

An unfortunately common mistake in barrier systems (Laminar
Flow Unit, Biological Safety Cabinet, Restricted Access Barrier
System, Isolator) design and integration into the cleanroom is
the position of the barrier system inlet in close proximity to the
HEPA filter inlet with relation to the surrounding cleanroom (see
Figure 7). This creates a challenge by reducing the contamination
control effect of the surrounding cleanroom.

Figure 7 HEPA filtered air from Grade B is sucked

into the RABS inlet

Drawing generated by Microrite

RABS air inlet creates upward air flow, overpowering

cleanroom air return system
Another common mistake in RABS and isolator design is the inlet
air to the barrier system provides an upward movement of air (see
Figure 8).
 Air Flow Visualization 157
Figure 8 RABS air inlet creates upward air flow

Drawing generated by Microrite

RABS cannot maintain first air over product contact

surfaces with door open
This condition is amplified when the RABS or isolator doors are
opened. During open door situations, first air does not make it to
product contact surfaces (see Figure 9). In unidirectional flow clean-
rooms, deviant air takes the form of eddy currents and turbulence;
these are considered a channel or reservoir for contamination.

Unidirectional flow versus laminar flow

Proper terminology should be used when discussing air flow in
cleanrooms. The use of “laminar air flow” when describing air
flow in cleanrooms was incorrectly applied in early cleanroom
specifications and standards (Fed-Std 209). However, this was
corrected in 1999 when unidirectional air flow replaced laminar air
flow in the International Cleanroom standards (ISO 14644 series).
Laminar flow describes air flow conditions that are impossible to
maintain in an open cleanroom, whereas unidirectional flow is a
more descriptive term (see Figure 10).
158 Cleanroom Contamination Prevention and Control
Figure 9 RABS cannot maintain first air over product contact
surfaces with door open

Drawing generated by Microrite

Figure 10 Laminar vs unidirectional airflow

Drawing generated by Microrite

 Air Flow Visualization 159
Figure 11 How the work surface itself causes air patterns
and velocities to change

WHY PERFORM AIR FLOW VISUALIZATION STUDIES?

Aside from the regulatory requirements, air flow visualization is a
useful tool for assessing risk of contamination from personnel and
processing.

Personnel, equipment and material flow can influence airflow

and affect contamination levels in the most well designed and
integrated cleanrooms and barrier systems.

Often air flow visualization studies are only performed

once at the initial qualification, only with the intent of meeting a
regulatory requirement. However, this limited approach does not
take advantage of these studies as a contamination control tool that
can help reduce the risk of contamination to products and product
contact surfaces.

Air flow visualization studies when used as part of a holistic

and comprehensive strategy, can assist in reducing the risk of
contamination by assessing the physical contamination control
effect of air flow when utilized in:
160 Cleanroom Contamination Prevention and Control

• Cleanroom qualification for both unidirectional and non-

unidirectional flow cleanrooms.
• Barrier system FAT/SAT qualification.
• Pass-through and air lock qualification.
• Optimization of cleanroom and barrier systems integration.
• Optimization of operator movements, standing positions,
equipment and material handling.
• Selection of environmental monitoring locations.
• Troubleshooting contamination issues and finding sources of air
patterns that may act as a channel or reservoir for contamination.
• Providing a training resource for operating personnel as a pro-
active approach to contamination control.

Air flow visualization studies provide a visual representation of

the contamination control effect of air flow patterns in cleanrooms
and controlled environments. This information is crucial when
evaluating the overall cleanroom/manufacturing area’s ability to
provide the desired level of contamination control. These studies
can be performed as an engineering tool prior to qualification, later
as part of qualification and repeated on a periodic basis as part of
the cleanroom requalification. Conducting air flow visualization
during cleanroom re-qualification is extremely important because
even successfully qualified systems can be compromised by poor
operational, maintenance, or personnel practices.

In aseptic operations, the characterization of air patterns

provides the most comprehensive overall representation of a
facilities contamination control strategy by allowing for the
evaluation of air patterns during simulations of operations.

From an overall contamination control standpoint, these

studies are as important as aseptic process simulations (media fills).
Where APS allows for the microbial evaluation of processing steps,
interventions and environmental monitoring, air flow visualization
 Air Flow Visualization 161

allows the evaluation of the overall contamination control effect

of air flow during similar simulations. This is a useful tool to
fine-tune operator movements and equipment setup challenges
prior to performing media fills. Because video documentation is
recommended, these studies can be used for training purposes to
optimize operator aseptic behavior.

WHAT ARE THE BENEFITS FROM

AIR FLOW VISUALIZATION?
Utilizing air flow visualization studies as part of a holistic and
comprehensive contamination control strategy, companies can
assess the physical contamination control effect of their cleanrooms.

• Evaluate if there is sufficient mixing of HEPA filtered air in non-

unidirectional flow cleanrooms.

• Determine if air returns are blocked or ineffectively located. Air

returns often are blocked by the placement of equipment, shelves
or carts – these may be permanent or temporary obstructions.
Objects placed in front of air returns can significantly hinder a
cleanroom’s ability to provide optimum contamination control.
Occasionally objects (e.g., cleanroom wipers, gloves, settle
plates, packaging, cleanroom paper) get sucked into open
returns (returns without grills or perforations) and clog the air
flow path upstream of the air return.

• Air flow visualization is useful in the evaluation of material

placement in relationship to contamination control. As air returns
are often placed on the perimeter of the cleanroom, products and
materials will often be placed out of the way and unfortunately in
front of an air return. The placement of clean or sterile products
in front of an air return should be considered poor practice,
as air returns represent the highest concentration of airborne
contamination (particles/microbes) in the operational cleanroom.
This can be visually represented via air flow visualization as
all tracer particles (smoke) released into the cleanroom will be
162 Cleanroom Contamination Prevention and Control

eventually removed from the cleanroom via the air return. It is

important that manufacturing and maintenance personnel are
aware of this phenomenon and avoid placing anything in front of
an air return. Video documentation of this effect can be included
as part of a cleanroom training program.

• Evaluate if there is a sweeping action over and away from the

product in unidirectional flow cleanrooms. (This is the most
significant part of cleanroom air flow, the “contamination
control effect”.)

• Identification of deviant air patterns such as excessive turbulence

or eddy currents that can act as a channel or reservoir for
contamination (e.g., from a particle generating person, operation,
machine or adjoining area of lower classification or Grade).

• Evaluate any heat sources that may impact air patterns. (Air
movement from convection can impair a cleanroom’s ability to
provide suitable contamination control.) For this reason, heat
sources (e.g., autoclaves and sterilization equipment) should
be at operational temperature during air flow visualization
studies. Additional scrutiny of exhaust ports or cooling fans of
equipment is suggested.

• Evaluate the effects on air patterns from mobile equipment,

transport carts, transfer RABS, transfer isolators during
simulations.

• Evaluate the effects of door openings/closings on air flow in

critical areas.

• Evaluate the effects of pass-throughs and air locks on air flow in

critical areas.

• Evaluate the contamination control effect of air flow patterns in

the cleanroom, barrier systems and the interface between areas
of different classification/Grades, e.g., Grade A RABS with a
Grade B background.
 Air Flow Visualization 163

• Evaluate operator movements, positions and attitudes in respect

to maintaining the contamination control effect of clean air flow
during simulations of normal operations.

• Evaluate operator movements, positions and attitudes in respect

to maintaining the contamination control effect of clean air flow
during simulations of high-risk operations such as equipment
set-up and assembly of product contact components, inherent
interventions, corrective interventions and environmental
monitoring operations.

WHAT ARE THE CONSEQUENCES OF IMPROPERLY

CONDUCTED AIR FLOW VISUALIZATION STUDIES?
Improperly conducted smoke studies can lead to the qualification
of cleanroom/barrier systems that are unsuitable for aseptic or low
bioburden operations. Because these studies are improperly carried
out, the resulting conclusions ignore excessive turbulence, air patterns
that act as a contamination transport mechanism into the critical area
or air patterns that act as a reservoir for contamination, e.g., eddy
currents or vortexes in the gap between HEPA filters. As the risk of
contamination is not identified, it cannot be addressed in the risk
management system and results in a variety of problems such as:
• Failed media fills.
• Contaminated products.
• Customer complaints.
• 483 observations.
• Warning letters.
• Import alerts.
• and more dire consequences.

The smoke study technique, tracer particle properties, camera angles

and documentation could be the assignable cause for not qualifying
airflows correctly.
164 Cleanroom Contamination Prevention and Control

Air flow visualization is often approached as a regulatory

requirement and not as part of a comprehensive contamination
control strategy.

The reason smoke studies are getting a lot of scrutiny is because

the FDA and European inspectors are becoming more aware of air
flow and cleanroom/barrier system design and integration flaws.
Additionally, data integrity issues resulting from smoke studies that
incorrectly qualify cleanroom or barrier system designs have led
companies to falsifying APS (media fill) results and environmental
monitoring data.

In some instances, smoke studies are manipulated in such a

manner as to provide a favorable outcome, even when it is apparent
there are cleanroom/barrier design flaws.

Camera angles and the position of the tracer particle manifold can
create an “optical illusion” that unidirectional air is present in critical
areas, when actually the smoke manifold is not over the critical area.

Certain types of tracer particles that create a heavier than air fog
have also been used to mask eddy currents, excessive turbulence
and dead spaces as a common form of manipulating smoke studies
in order to reach a more favorable conclusion.

Case histories
Lyophilization transfer trolley creates excessive turbulence
when docking to the RABS and lyophilizer
A liquid filling line received an FDA 483 observation after inspectors
reviewed their smoke study videos of the transfer of vials to the
lyophilizer. The company used a mobile (battery powered) HEPA
trolley (cart) to maintain a Grade A (ISO Class 5) unidirectional flow
environment during the transfer of partially stoppered vials to the
lyophilizer. This transfer trolley had a powerful fan that created an
excessive amount of turbulence when docked at the Grade A filling
RABS enclosure as well docked at the lyophilizer. The design of the
 Air Flow Visualization 165

transfer trolley was not considered in the overall qualification of the

manufacturing line.

False positives in microbial sampling from a bioreactor due

to HMI cooling fan
A pharmaceutical company with an ISO Class 7 cleanroom had an
aseptic connection located under a HEPA filter using cleanroom
curtains. The bioreactor (where the sample port was located) was
controlled by an HMI Human Machine Interface (HMI) computer.
The HMI was enclosed in a stainless-steel box with a cooling fan. The
HMI was positioned on an articulating swing arm. The preferred
operational position of the HMI on the articulating swing arm was
such that the cooling fan was blowing unfiltered air in the direction
of the aseptic connection.

The smoke study revealed that the HMI fan was capable of
overpowering the HEPA filter above the aseptic connection as
smoke released in front of the HMI exhaust would be detected by
a particle counter with its isokinetic sample port located next to the
aseptic connection. To note the aseptic connection was located under
a HEPA filer and surrounded by cleanroom curtains.

The HMI was upgraded to a more cleanroom compatible model

(without a cooling fan) and the aseptic connection environment was
significantly improved.

New installation, failed media fills

A new Contract Manufacturing Organization’s (CMO) facility
installed a new liquid filling line utilizing open passive RABS,
underneath a grid of HEPA filters in the ceiling. The cleanroom
qualification was completed inclusive of air flow visualization.
Unfortunately, the RABS loading ports had excessively turbulent air
flow, something the initial air flow visualization did not detect. This
air flow visualization study was conducted utilizing a heavier than
air “cleanroom fog” that gave the false impression that the loading
ports for sterilized materials had true unidirectional flow.
166 Cleanroom Contamination Prevention and Control

The failed media fill led to an investigation utilizing a neutrally

buoyant fog that clearly demonstrated the excessive turbulence in
the loading ports. The RABS was modified to provide unidirectional
air flow, smoke studies were repeated, and the following media fills
passed.

Data integrity and smoke studies

A Contract Manufacturing Organization (CMO) was cited for data
integrity related to environmental monitoring data as well as media
fill results. To an experienced inspector the cleanroom design and
RABS installation were not suitable for aseptic operations as the
cleanroom was too small for the operations being carried out. A
Laminar Air Flow (LAF) unit was suspended under the cleanroom
ceiling and a filling machine was placed in custom RABS enclosure.
The addition of the RABS enclosure made the operational space
for the personnel too small. To the educated eye, deviant air flow
would be apparent in any smoke study carried out. Properly
conducted viable and non-viable monitoring as well as media fills
should have detected at least an occasional excursion. However, all
environmental monitoring data and media fill data indicated nearly
perfect conditions.

This company-wide data integrity violation started when

the customer discovered that by switching the LAF unit off and
conducting an air flow visualization with a heavier than air
“cleanroom fog” unidirectional air flow could be demonstrated for
video documentation purposes. The real environmental monitoring
routinely had excursions and media fills routinely had growth,
however data were falsified in order to meet customer’s demands.

WHAT ARE THE INTERNATIONAL STANDARDS?

The International Organization for Standardization (ISO)
established a basic set of guidelines and test criteria for the design
and qualification of general cleanroom operations. The ISO 14644
 Air Flow Visualization 167

series of standards titled “Cleanrooms and associated controlled

environments” provides only a basic set of guidelines, equipment
specifications and test criteria regarding the design and qualification
of general cleanroom operations.

Though air flow direction is listed as an optional test (as the

standard is for all industries) air flow visualization (smoke studies)
are a regulatory expectation in pharmaceutical manufacturing and
hospital formulation facilities.

Air flow visualization testing is addressed in the international

cleanroom standard ISO 14644-3:2005 Part 3 Test Methods section 4.2
Principle. 4.2.5 (ISO, 2005)) Airflow direction test and visualization:

“The purpose of this test is to confirm either the airflow direction

or airflow pattern or both in regard to the design and performance
specifications. If required, spatial characteristics of airflow in the
installation may also be confirmed.”

The standard introduces the tracer particle injection method: “The

test is carried out by observation or imaging of the behavior of Tracer
Particles”. In addition, the standard refers to the methods from which
the tracer particles may be generated: “The tracer particles can be
generated from materials such as de-ionized (DI) water, sprayed or
chemically generated alcohol/glycol etc.”

Additional caution is provided related to the tracer particles

“size and the effects of gravity:

“The desired size of droplets should be considered when selecting

the droplet generation method. Droplets should be large enough to
be detected with the available image processing techniques, but not
so large that gravitational or other effects will result in their motion
diverging from that of the airflow being observed.”
168 Cleanroom Contamination Prevention and Control

WHAT ARE THE REGULATORY REQUIREMENTS,

GUIDELINES OR EXPECTATIONS?
Medical product cleanrooms for sterile or aseptic processing require
additional design, qualification and operational considerations than
those detailed in the ISO 14644 series of standards. The require­
ments for cleanrooms used in the manufacturing, formulation and
handling of medical products extend beyond the requirements of
those cleanrooms used in other industries, i.e., microelectronics,
aerospace. For example, ISO Class 5 cleanrooms used in the manu­
facturing or handling of sterile or low bioburden products look
different and are operated differently than ISO Class 5 cleanrooms
used in producing microelectronics. Because of these differences,
additional guidance is required in designing, qualifying, operating
and monitoring medical product cleanrooms.

Though considered an optional test for general cleanroom

installations per ISO 14644-3 (ISO, 2005), air flow visualization testing
with video documentation is an expected test by pharmaceutical
inspection authorities and is also referred to as “smoke study” in
FDA, PIC/S, WHO and USP guidance documents.

As there is no specific requirement in the FDA GMP regulations,

the FDA has issued FDA Form 483 observations and warning letters
citing the lack of smoke testing or inadequate smoke testing in
critical (sterile or aseptic) processing areas. These observations often
reference the following sections of 21 Code of Federal Regulations
(CFR) Part 211, Current Good Manufacturing Practice for Finished
Pharmaceuticals.

Subpart C – Buildings and Facilities

Sec. 211.42 Design and construction features.

(10) Aseptic processing, which includes as appropriate:

(i) Floors, walls, and ceilings of smooth, hard surfaces that are easily
cleanable;

(ii) Temperature and humidity controls;

 Air Flow Visualization 169
(iii) An air supply filtered through high-efficiency particulate air filters
under positive pressure, regardless of whether flow is laminar or
nonlaminar;

(iv) A system for monitoring environmental conditions;

(v) A system for cleaning and disinfecting the room and equipment to
produce aseptic conditions;

(vi) A system for maintaining any equipment used to control the

aseptic conditions.

Sec. 211.113(b) Appropriate written procedures, designed to prevent

microbiological contamination of drug products purporting to be
sterile, shall be established and followed. Such procedures shall
include validation of any sterilization process.

Sec. 211.63 Equipment used in the manufacture, processing, packing,

or holding of a drug product shall be of appropriate design, adequate
size, and suitably located to facilitate operations for its intended use
and for its cleaning and maintenance.

21 CFR Part 211.113(b) is generally cited when the video shows

operator activities are contributing to turbulence or the true air flow
cannot be demonstrated based on the poor placement of the smoke
source and camera angle.

21 CFR Part 211.63 is generally cited when the video shows the line
design/equipment placements are contributing factors to turbulence.

As the 21 CFR regulations are somewhat vague, additional guidance

from the FDA in the form the following document is frequently
quoted in inspection reports and warning letters.

The US FDA “Guidance for Industry Sterile Drug Products

Produced by Aseptic Processing Current Good Manufacturing
Practice” (FDA, 2004):

Page 6: “HEPA-filtered air should be supplied in critical areas at a

velocity sufficient to sweep particles away from the filling/closing area
and maintain unidirectional airflow during operations. The velocity
170 Cleanroom Contamination Prevention and Control
parameters established for each processing line should be justified and
appropriate to maintain unidirectional airflow and air quality under
dynamic conditions within the critical area.

Proper design and control prevent turbulence and stagnant air in the
critical area. Once relevant parameters are established, it is crucial
that airflow patterns be evaluated for turbulence or eddy currents that
can act as a channel or reservoir for air contaminants (e.g., from an
adjoining lower classified area). In situ air pattern analysis should be
conducted at the critical area to demonstrate unidirectional airflow
and sweeping action over and away from the product under dynamic
conditions.

The studies should be well documented with written conclusions

and include evaluation of the impact of aseptic manipulations (e.g.,
interventions) and equipment design.

Videotape or other recording mechanisms have been found to be useful

aides in assessing airflow initially as well as facilitating evaluation of
subsequent equipment configuration changes. It is important to note
that even successfully qualified systems can be compromised by poor
operational, maintenance, or personnel practices.”

In 2020, technological advances in camera resolution and video

storage should be considered when developing air flow visualization
procedures. Video tapes and low-resolution video recordings are
difficult to justify when applying “cGMP” to a company’s risk-based
approach to contamination control. Compact flash memory in USB
and SD formats as well as cloud-based storage options should be
considered when developing air flow visualization procedures.

GMP Annex 1 2008: Both the EU (EU GMP, 2008) and PIC/S
(PICS/GMP Guide) versions provide additional information that
should be considered in cleanroom design and operations as it
applies to air flow visualization. European Commission Eudralux
Volume 4, Annex 1 (2008) and PIC/S GMP Guide (PE 009-11) Annex
1 Clause 3, 53, 54:
 Air Flow Visualization 171
Paragraph 3. Clean areas for the manufacture of sterile products are
classified according to the required characteristics of the environment.
Each manufacturing operation requires an appropriate environmental
cleanliness level in the operational state in order to minimize the risks
of particulate or microbial contamination of the product or materials
being handled.

In order to meet “in operation” conditions these areas should be

designed to reach certain specified air-cleanliness levels in the “at
rest” occupancy state. The “at-rest” state is the condition where the
installation is installed and operating, complete with production
equipment but with no operating personnel present. The “in operation”
state is the condition where the installation is functioning in the defined
operating mode with the specified number of personnel working.

The “in operation” and “at rest” states should be defined for each clean
room or suite of clean rooms.

For the manufacture of sterile medicinal products 4 grades can be

distinguished.

Grade A: The local zone for high risk operations, e.g. filling zone,
stopper bowls, open ampoules and vials, making aseptic connections.
Normally such conditions are provided by a laminar air flow
workstation. Laminar air flow systems should provide a homogeneous
air speed in a range of 0.36–0.54 m/s (guidance value) at the working
position in open clean room applications. The maintenance of
laminarity should be demonstrated and validated.

Paragraph 53: A filtered air supply should maintain a positive pressure

and an air flow relative to surrounding areas of a lower grade under all
operational conditions and should flush the area effectively.

Adjacent rooms of different grades should have a pressure differential

of 10–15 pascals (guidance values).

Particular attention should be paid to the protection of the zone of

greatest risk, that is, the immediate environment to which a product
and cleaned components which contact the product are exposed.
172 Cleanroom Contamination Prevention and Control
The various recommendations regarding air supplies and pressure
differentials may need to be modified where it becomes necessary
to contain some materials, e.g. pathogenic, highly toxic, radioactive
or live viral or bacterial materials or products. Decontamination of
facilities and treatment of air leaving a clean area may be necessary for
some operations.

Paragraph 54: It should be demonstrated that air-flow patterns do not

present a contamination risk, e.g. care should be taken to ensure that
air flows do not distribute particles from a particle-generating person,
operation or machine to a zone of higher product risk.”

As of the time of this writing, GMP annex 1 is up for revision.

Additional guidance related to smoke studies as well as the

introduction of the L-R (Ljundqvist and Reinmüller) Method
(Ljundqvist and Reinmüller, 1993) are addressed in USP <1116>: USP
Section 1116: Microbiological Control and Monitoring of Aseptic
Processing Environments.

“Manufacturers should maintain a predominantly unidirectional flow

of air (either vertical or horizontal) in a staffed Class 5 clean room
environment, particularly when products, product containers, and
closures are exposed.

In the evaluation of air movement within a clean room, studying

airflow visually by smoke studies or other suitable means is probably
more useful than using absolute measures of air-flow velocity and
change rates.

Physical Evaluation of Contamination Control Effectiveness:

ISO 14644 series in order to meet their design classification
requirements. The design, construction, and operation of clean rooms
vary greatly, so it is difficult to generalize requirements for parameters
such as filter integrity, air velocity, air patterns, air changes, and
pressure differential. In particularly critical applications such as
aseptic processing, a structured approach to physical risk assessment
may be appropriate.
 Air Flow Visualization 173
One such method has been developed by Ljundqvist and Reinmuller.
This method, known as the L-R method, challenges the air ventilation
system by evaluating both airflow and the ability of an environment
to dilute and remove air-borne particles. In the L-R method, a smoke
generator allows analysts to visualize the air movements throughout
a clean room or a controlled environment, including vortices or
turbulent zones, and the airflow pattern can be fine-tuned to minimize
these undesirable effects. Following visual optimization of airflow,
particulate matter is generated close to the critical zone and sterile
field. This evaluation is done under simulated production conditions
but with equipment and personnel in place. This type of test can
also be used to evaluate the ability of RABS and isolator systems,
particularly around product exit ports in these systems, to resist the
effects of contamination.

Visual evaluation of air movement within clean rooms is a subjective

process. Complete elimination of turbulence or vortices is not possible
in operating cleanrooms that contain personnel and equipment. Air
visualization is simply one step in the effort to optimize cleanroom
operations and is not a definitive pass/fail test, because acceptable or
unacceptable conditions are not readily definable.

Proper testing and optimization of the physical characteristics of the

cleanroom, RABS, or isolator are essential before implementation
of the microbiological monitoring program. Assurance that the
cleanroom, RABS, or isolator is in compliance with its predetermined
engineering specifications provides confidence that the ability of the
facility systems and operating practices to control the bioburden and
nonviable particulate matter are appropriate for the intended use. These
tests should be repeated during routine certification of the cleanroom
or advanced aseptic processing systems, and whenever significant
changes are made to the operation, such as personnel flow, equipment
operation, material flow, air-handling systems, or equipment layout.”

WHO GMP for Sterile Pharmaceutical Products Working Document

Clause 4.3:

“The uniformity and effectiveness of the unidirectional airflow shall be

demonstrated by undertaking airflow visualization tests.”
174 Cleanroom Contamination Prevention and Control

Pharmaceutical compounding
As the compounding of pharmaceuticals is different than
manufacturing, different rules and guidance apply. For the
compounding of sterile preparations and medications, USP <797>
has specific requirements related to smoke studies during dynamic
conditions. The USP requirements are listed below. Pharmaceutical
Compounding Sterile Preparations USP Section <797>:

“Dynamic airflow smoke pattern test: Smoke pattern tests must be

performed for each PEC during dynamic operating conditions to
demonstrate unidirectional airflow and sweeping action over and
away from the preparation(s).

Placement and movement of Materials: Only equipment necessary

for performing compounding activities is permitted in the PEC.
Proper placement of equipment in a PEC must be initially verified by a
dynamic airflow smoke pattern test to demonstrate minimal disruption
in airflow. The dynamic airflow smoke pattern test must be repeated if
equipment is placed in a different location.

Dynamic airflow smoke pattern test: Smoke pattern tests must be

performed for each PEC during dynamic operating conditions to
demonstrate unidirectional airflow and sweeping action over and
away from the preparation(s).The number of personnel present in each
PEC and SEC during total particle count tests and dynamic airflow
smoke pattern tests must be documented.

Dynamic airflow smoke pattern test: A PEC test in which a visible

source of smoke, which is neutrally buoyant, is used to observe air
patterns within the unidirectional space (i.e., the DCA) under dynamic
operating conditions (see Dynamic operating conditions). This test is
not appropriate for ISO Class 7 or ISO Class 8 cleanrooms that do not
have unidirectional airflow (see Visual smoke study).

Dynamic operating conditions: Conditions in the compounding area

in which operating personnel are present and simulating or performing
compounding. The conditions should reflect the largest number of
personnel and highest complexity of compounding expected during
routine operations as determined by the designated person(s).
 Air Flow Visualization 175
Visual smoke study: A test, used in ISO Class 7 and ISO Class 8 rooms
that do not have unidirectional airflow, in which a visible source of
smoke, which is neutrally buoyant, is used to verify an absence of
stagnant airflow where particulates can accumulate. This test does not
need to be performed under dynamic operating conditions and is not
appropriate for PECs (see Dynamic airflow smoke pattern test).

A dynamic airflow smoke pattern test must be performed in the PEC

under dynamic operating conditions initially and at least every 6
months to ensure that:

1) the RABS is properly integrated into the facility and

2) compounders understand how to utilize the unidirectional airflow

to maintain first air in the DCA.”

For the testing of Pharmaceutical Compounding cleanrooms (per

USP <797>) the Controlled Environment Testing Association (CETA)
has created additional guidance. CETA Certification Guide for Sterile
Compounding Facilities CAG-003-2006 -13 Revised May 20, 2015:

“13.3 A visible source of smoke such as a glycol-based fog generator

or ventilation smoke tube is used to observe air patterns within the
unidirectional space.

Smoke is generated directly downstream of the diffuser and then

observed as it flows across the critical site and the direct compounding
area (DCA) and to a return or out of the critical area. Air exiting the
critical area should not re-enter.

13.5 Water based fog generators such as CO2 and liquid nitrogen create
a fog that is heavier than air and do not always provide for an accurate
representation of the actual air patterns.

The smoke source should be as close to neutrally buoyant as possible.

For example, when generating the fog in an area with no detectable
airflow, it should not “fall out” or “drop”.

Fog streams that are heavier than air may not detect updrafts and
turbulence that are detected with a generally neutral buoyant
detection stream.”
176 Cleanroom Contamination Prevention and Control

REGULATORS COMMENTS ON SMOKE STUDIES

As smoke studies are required by FDA and international bodies, the
review of these studies is a common practice during inspections.
The following are quotes from FDA 483 observations or Warning
letters.

“During the airflow analysis (smoke study) of aseptic connections

on your equipment inside the laminar air flow (LAF) ISO-5 area, our
investigator identified air flow disturbances and turbulence. Under
dynamic conditions, air did not sufficiently sweep across and away
from sterile connections, so the sterility of any product processed
under these conditions could be compromised. Furthermore, in our
review of the smoke study, we identified multiple aseptic technique
breaches during aseptic connection of the equipment.

Your equipment design and aseptic processing operator competencies

appear to contribute to the lack of unidirectionality.”

“The sterile filling line for injectable products lacks unidirectional

airflow in the ISO 5 aseptic filling zone. The RABS airflow in the filling
zone is not sufficiently robust to protect the sterile injectable product
during interventions involving operator entry into the aseptic filling
line. Smoke studies demonstrated that the filling line design permits
turbulence above and below open vials. Opening the enclosure
significantly disrupts airflow. This turbulent air in the aseptic filling
zone poses a significant contamination hazard.”

“You have not performed smoke studies under dynamic conditions

to demonstrate and assure proper air flow patterns in the ISO 5
environment, during normal working conditions, smoke studies were
not performed for the worksurfaces where aseptic operations are
carried out.”

“Aseptic Manipulations are performed in an area where the

unidirectional movement of air in the ISO 5 area is disrupted.

Specifically, smoke studies completed in laminar airflow workbench

demonstrated that air refluxes around the control panel and where the
sterile connections are made.”
 Air Flow Visualization 177

Frequent inspector’s observations often include:

• “Smoke Studies not conducted during Dynamic conditions

simulating operations with equipment running, filling and
stoppering.”

• “Only Grade A areas and not support Grade B areas tested.”

• “Only one angle recorded during dynamic smoke studies,

operator’s body blocked the camera angle during part of the
video…”

• “Smoke Study Video not available for inspectors.”

• “Dynamic Smoke Study Video does not reflect actual operations

as indicated in Media Fill.”

• “Dynamic Smoke Study does not include all operations, Normal

and Unusual interventions.”

• “Smoke Generator does not generate adequate smoke to evaluate

the aseptic process.”

• “Smoke Generator generates too much smoke.”

• “Smoke Studies do not fully demonstrate air flow movement

away from work surfaces during interventions.”

AIR FLOW VISUALIZATION APPLICATIONS

In addition to regulatory expectations, air flow visualization is
a useful engineering and validation tool that can be used for the
following applications:

Cleanroom qualification
(For unidirectional and non-unidirectional flow cleanrooms.) In the
qualification of cleanrooms, mapping of air flow patterns is used for
confirming design and performance specifications.
178 Cleanroom Contamination Prevention and Control

Equally important is the identification of areas where air flow

patterns have a lesser contamination control effect (e.g., doors, pass-
throughs, spaces between HEPA filters and areas with equipment
exhaust fans).

• Unidirectional airflow should exist across the entire Grade A

(ISO Class 5) area. Any deviant air such as excessive turbulence,
eddy currents or vortexes should be identified and addressed
in the risk assessment and evaluated in the contamination
control strategy. When possible, optimization/modification of
the cleanroom should occur. If a physical modification is not
possible, additional environmental monitoring scrutiny of these
areas must be undertaken.

• For non-unidirectional air flow cleanrooms, it should be

demonstrated that HEPA filtered air entering the cleanroom
effectively mixes with room air and exhausts via low wall
air returns. Important for medical product cleanrooms is the
identification of any areas of concern (such as equipment cooling
fans, heat sources, pneumatics, product handling robotics
or areas with poor mixing or no air movement). These areas
once identified, must be addressed in the risk assessment and
evaluated in the contamination control strategy.

• For non-unidirectional air flow areas that support critical

operations, it is important that air flow patterns from equipment
cooling fans, heat sources, pneumatics, product handling
robotics do not pose a contamination risk by washing over
operators or components that may enter critical areas or are in
close proximity to critical areas.

Barrier system FAT/SAT qualification

In the qualification of barrier systems (e.g., RABS, isolators) it is
important that unidirectional air flow is specified and demonstrated
as part for the FAT/SAT qualification, prior to performing official
in situ air pattern analysis. Too often poorly designed RABS and
 Air Flow Visualization 179

isolators already integrated into a production cleanroom have been

unable to provide a suitable environment for aseptic processing
due to excessive turbulence and eddy currents caused by large gaps
between HEPA filters.

• FAT: the demonstration of unidirectional flow and a con­

tamination control effect for all use-case conditions must be
performed prior to equipment acceptance. Especially important
is the demonstration of open-door conditions for RABS and
isolators.

• SAT: the ability of the barrier system to prevent outside air from
entering is critical to establishing a suitable environment for
aseptic or sterile operations. Large product contact components
such as stopper bowls and hoppers must be appropriately
sterilized and transferred into the filling or processing
equipment. As these items are too large to be transferred via
product transport ports, it is imperative that during equipment
set-up, outside air (Grade B for RABS, Grade C for isolators)
cannot enter the Grade A barrier system’s environment.

Pass-through and air lock qualification

Pass-throughs and air locks should be designed and used to provide
physical separation and to minimize microbial and particulate
contamination of the different areas.

The qualification of pass-through and airlocks should utilize

air flow visualization as a tool to evaluate these devices ability to
act as an air lock. Tracer particles (smoke) should not leak from
the pass-through or air lock into the critical area. Too often pass-
through gaskets are not integral, damaged or missing. The use of
air flow visualization provides a useful pass-through leak test that
effectively tests the “air lock” principle.

Active or dynamic pass-throughs (with fan powered HEPA filter

modules) can be evaluated for the effectiveness of the air exchange,
180 Cleanroom Contamination Prevention and Control

or how quickly the pass-through flushes itself with clean air.

Evaluation of pass-through air flow patterns should be performed
simulating operations with the maximum material or equipment
conditions.

Optimization of cleanroom and

barrier systems integration
Often the overall contamination control effect of sterile or aseptic
processing cleanrooms is compromised by poor integration of barrier
systems into cleanrooms. After the cleanroom and barrier systems
have demonstrated suitable air patterns as part of their individual
qualifications, air flow visualization should be conducted in order to
evaluate the air flow patterns related to the integration of the barrier
system within the cleanroom.

• Air patterns inside of barrier systems must not be influenced by

external activities such as the opening and closing of cleanroom
doors, or the movement of personnel in the surrounding
environment (see Case Histories, above).

• Air patterns inside of barrier systems must not be influenced by

the connection of mobile HEPA transfer carts, transfer RABS or
transfer isolators (see Case Histories, above).

• The evaluation of air patterns and the interface between the

cleanroom and barrier system must be evaluated in all possible
scenarios, including the opening and closing of doors. Important
consideration related to the placement of air returns and
exhaust system’s impact on air patterns should be evaluated in
relationship to contamination risk.

Optimization of operator movements and positions

As operator movements and positions can influence air patterns,
characterization of these movements as well as standing positions
can be assessed in terms of air pattern analysis. Air flow visualization
 Air Flow Visualization 181

testing as an engineering tool can help optimize operator movements

as well as positions in order to achieve improved air flow and
increased contamination control.

Training tool
Additionally, videos from air pattern analysis can providing training
material as a method of critiquing operator behavior in relationship
to air flow patterns, particularly while performing aseptic techniques,
interventions and environmental monitoring.

Establishing risk based environmental

monitoring locations
When establishing environmental monitoring locations and
methods, the review of air flow visualization studies should assist
in the selection of risk based environmental monitoring locations for
conducting viable and non-viable particle monitoring. Additionally,
this information can assist when selecting locations for cleanroom
classification testing as well.

Frequency of air flow visualization testing

and cleanroom re-qualification
The frequency of performing air flow visualization studies is depen-
dent upon the type of facility as well as the operations carried out.

Air flow visualization studies should be repeated during routine

certification/re-qualification of the cleanroom or advanced aseptic
pro­cessing system and whenever significant changes are made to the
operation, such as changes to personnel flow, equipment operation,
material flow, air handling systems or equipment layout. It is im­portant
to note that even successfully qualified systems can be compromised
by poor operational, maintenance, or personnel practices.
182 Cleanroom Contamination Prevention and Control

In the re-qualification of cleanrooms, it is important to compare

studies historically to asses any differences from the original air flow
visualization study.

Pharmaceutical manufacturing air flow

visualization testing frequency
The maximum interval for repeating air flow visualization studies
for pharmaceutical manufacturing is not defined in regulatory
documents however, the first edition of ISO 14644-2 (2000) indicated
repeating this test at a maximum interval of 24 months. This was
changed in the second edition (ISO 14644-2 (2015) to be based upon
risk assessment however many manufacturers are repeating the test
between 12 and 24 months.

Pharmaceutical compounding air flow

visualization testing frequency
The maximum interval for repeating air flow visualization studies
for Sterile Pharmaceutical Compounding facilities (per USP <797>)
is six months. The increased frequency is meant to address the
fluid nature of compounding as well as the transportable nature
of equipment, tables and chairs often utilized in pharmaceutical
compounding facilities.

TYPES OF AIR FLOW VISUALIZATION

Cleanroom air flow visualization can be undertaken to characterize
air flow patterns in several different types of studies.

• Investigative.

• Engineering.

• Static.

• Dynamic.
 Air Flow Visualization 183

Investigative air flow visualization

Investigative air flow visualization studies are used for identifying
specific contamination control issues or as part of a cleanroom audit.
This type of testing is a useful tool in troubleshooting contamination
and cross contamination problems. Investigative smoke studies can
detect air flow patterns that could act as a transport mechanism or
as a reservoir for contamination. To avoid problems prior to media
fills, these studies should be done prior to conducting formal and
documented “Dynamic in situ air pattern analysis” in order to detect
and address problems before qualification testing starts.

Engineering air flow visualizations

Engineering air flow visualization studies are used as an engineering
tool in order to characterize the cleanroom air flows as well as the
cleanroom and if applicable any barrier system integration. Of
particular importance is the interface between different clean zones
such as between Grade A and Grade B zones in aseptic manufacturing.
Additionally, engineering air flow visualization is useful as part of a
physical risk assessment of the contamination control effectiveness
and should be used for establishing environmental monitoring
locations within the contamination control strategy. These types
of studies should be specified in equipment URS and required as
part of FAT and SAT testing of any barrier system (isolators, RABS).
Particular attention should be given to the interface between barrier
systems and the external environment. To avoid problems prior to
media fills, these studies should be done prior to conducting formal
and documented “Dynamic in situ air pattern analysis” in order to
detect and address problems before qualification testing starts.

Static air flow visualization studies

Static air flow visualization studies are used to characterize the
air flow patterns of the entire cleanroom and any associated clean
air devices, barriers systems (RABS, isolators or HEPA carts).
All doors, pass-throughs, conveyer belts and equipment that can
184 Cleanroom Contamination Prevention and Control

impact air patterns must be evaluated. The spatial relationship of

HEPA filter diffusers and air returns must be evaluated in terms of
overall contamination control effect of the area being tested. These
studies are a pre-requisite of “Dynamic in situ air pattern analysis”.
The logic is if a static AFV/smoke study reveals turbulence or eddy
currents or air moving from a less critical area to a critical area under
static conditions then we do not meet the requirements of proper
cleanroom air flow. Poor air flow under static conditions equates to
poor air flow under dynamic conditions.

“Dynamic in situ air pattern analysis”

(aka dynamic smoke studies)
Dynamic in situ air pattern analysis is used to characterize air
flow patterns during simulations of operations. Because these
studies are focused on the movements of people and equipment,
the overall con­tamination control effect of the cleanroom cannot be
evaluated only with dynamic air flow visualization studies. Prior to
conducting a “Dynamic in situ air pattern analysis” a static air flow
study must be performed. The FDA’s expectation (FDA, 2004) is a
“Dynamic in situ air-pattern analysis” which is used to demonstrate
unidirectional airflow and a sweeping action over and away from
the product under dynamic conditions. Multiple cameras may be
used to best reflect and document the air flow. This type of study is
often used as evidence of suitable air patterns as recommended by
regulatory authorities.

What is “Dynamic in situ air pattern analysis”?

As the words in situ implies “in the original, natural position;
undisturbed,” this is an important consideration when performing
air flow visualization studies. Having an additional test person
holding a smoke tube over the operator performing an intervention
does not represent the natural air flow situation. Manifolds and
fixtures should be utilized as much as possible to better provide
a more realistic simulation of interventions performed during
dynamic in situ air pattern analysis.
 Air Flow Visualization 185

SIMULATIONS OF ACTIVITIES DURING

AIR FLOW VISUALIZATION
Regarding the simulation of activities during dynamic air flow
visualization, the human operator is by far the greatest source of
microbial and particulate contamination during an aseptic process.
To demonstrate aseptic processing capability, air flow visualization
while performing process simulations is a regulatory expectation
and should include the simulation of: the transfer of materials
into the critical area, equipment set-up, assembly, environmental
monitoring in addition to all the inherent interventions (part of
the process) and corrective interventions (problem resolution) that
could occur during an aseptic filling process.

Interventions and risk in evaluating aseptic processing must

strive to avoid human interventions, and where they are unavoidable
to minimize their impact as much as possible. Inherent interventions
are activities that are integral parts of the aseptic process and every
batch. Corrective interventions are activities that rectify problems
and may not be a part of every batch.

Table 1 Examples of inherent and corrective interventions

Inherent Interventions Corrective Interventions

Line set-up and assembly Stopper jams
Replenishment of components Broken / fallen glass
Weight/volume checks and adjustments Defective seals on containers
Environmental monitoring Liquid leaks and spills
Breaks, lunch Other mechanical failures requiring manual
correction

PREPARATION FOR DYNAMIC SMOKE STUDIES

The importance of understanding how to perform these tests as
well as the nature of the dynamic testing in the critical environment
cannot be understated. Cleanroom testing professionals may or may
not understand the suitable methods for performing a dynamic
186 Cleanroom Contamination Prevention and Control

smoke study for a specific cleanroom. This is because they may not
have a clearly defined list of all personnel movement for all aseptic
operations.

Dynamic smoke studies require the simulation of production

activities. In order to correctly visualize airflow under dynamic
conditions, the cleanroom must be configured to simulate actual
operations. The equipment, product containers, vessels and transfer
materials must be in place. This also requires the actual personnel
ready to perform their operations. This may include support per­
sonnel that assist operators working in the critical environment, as
they may (due to their proximity to the critical environment) influence
air patterns as they support operators in the critical environment.

Setting up a dynamic smoke study with video evidence requires

the same complexity as setting up the filming of a small video or
movie production. Lighting, camera angles, (multiple camera
angles) as well as directing the operating personnel are all important
in performing a well-documented AFV/Smoke study.

Prior to performing any dynamic AFV/smoke study, it is

imperative to perform a static AFV/smoke study. The logic is: if a
static AFV/smoke study reveals turbulence or eddy currents or air
moving from a less critical area to a critical area under static conditions
then we do not meet the requirements of proper cleanroom air flow.
Poor air flow under static conditions equates to poor air flow under
dynamic conditions.

FIRE AND SMOKE DETECTION SYSTEMS

It is important to notify security and if required any external fire
monitoring agency that AFV/smoke studies are being conducted
in advance and just prior to commencing the testing. Not all fire/
smoke detection technologies are the same. Different technologies
used have different sensitivities to smoke/fog particle sizes.
 Air Flow Visualization 187

TRACER PARTICLE MANIFOLD POSITION

The position of the smoke manifold is extremely important. Figure
12 shows the correct position of the tracer particles manifold: just
below the HEPA diffuser. In Figure 12 we have unidirectional air
flow with first air sweeping over the product contact surfaces.

Figure 12 Correct position of the tracer particles manifold:

just below the HEPA diffuser

Drawing generated by Microrite

Figure 13 illustrates that when the RABS door is open, we no longer

have unidirectional flow inside the RABS. This is due to the poor
integration of the RABS into the cleanroom. This is problematic as
there is no longer unidirectional flow inside the RABS and first air is
not getting to product contact surfaces.

Figure 14 shows the incorrect position of the tracer particles

manifold: the manifold is placed here to hide the effect of the door
opening on the RABS internal air flow. Unidirectional air flow is not
maintained, and first air is not getting to product contact surfaces.
188 Cleanroom Contamination Prevention and Control
Figure 13 Lack of unidirectional airflow when the RABS door is open

Drawing generated by Microrite

Figure 14 Incorrect positioning of tracer particles manifold inside

RABS

Drawing generated by Microrite

Figure 15 shows the position of the tracer particles manifold for

testing a Grade B room. Because the HEPA supply air is very close
to the RABS inlet a “short circuit” is created. This is detrimental to
the overall contamination control effect of the facility.
 Air Flow Visualization 189
Figure 15 Position of the tracer particles manifold for testing a
Grade B room and a short circuit effect

Drawing generated by Microrite

TRACER PARTICLES
For accurate air flow visualization studies using the tracer particle
injection method, it is extremely important that the tracer particles
injected into the cleanroom air stream are as close to neutrally
buoyant as possible (Hinds, 1999).

Figure 16 Various cleanroom fogger technologies

and their settling velocity

Drawing generated by Microrite from Hinds (1999)

190 Cleanroom Contamination Prevention and Control

Certain types of “cleanroom foggers” utilizing ultrasonically

generated fog or liquid nitrogen and water create tracer particles too
large to be considered neutrally buoyant.

Tracer particle size

All particles are affected by gravity due to their mass. For tracer
particles used in AFV/smoke studies, the size/mass of the particles
used can influence the particles ability to remain in the air streams.
The smaller the tracer particle, the less mass it has, and the lower
settling velocity. The larger the tracer particle the greater the mass
and higher the settling velocity.

Larger sized tracer particles (≥5.0 µm) have too much mass and
are affected by gravity. Tracer particles affected by gravity are not
neutrally buoyant (in still air) and may not represent the actual air-
flow patterns as gravity will influence the tracer particles movement
once released into the cleanroom. These heavier particles sink to the
floor of the cleanroom in the absence of air flow and this can lead
to incorrect conclusions related to air pattern analysis. Sub-micron
tracer particles (≤1.0 µm) are less affected by gravity and are better
at representing the actual air flow patterns.

Duration (lifespan) of tracer particles

The material of the tracer particles should be such that it remains
visual from the time it is injected into the cleanroom air steam
until it exits that area being tested. Ideally the tracer particles
should remain visible until they are removed by the cleanrooms
exhaust (air return plenum). Tracer particles that remain visual
longer are useful in detecting air patterns in the perimeter that may
allow air to re-enter critical area after first exiting the critical area
due to poorly implemented cleanroom equipment integration or
personnel movement. Tracer particles that remain visual longer
are also useful in visually representing the mixing of air in non-
unidirectional flow cleanrooms and identifying any dead spaces
 Air Flow Visualization 191

that can act as a reservoir for particles. In addition, the impact of

cooling fans on various equipment can be visualized in relation to
the transmission of particles from possible contamination sources
within the cleanroom. Tracer particles that dissipate too rapidly
(e.g., ultrasonically generated water vapor) may not adequately
represent the actual cleanroom air flow patterns. Tracer particles
that dissipate too rapidly would not be able to evaluate mixing of air
in non-unidirectional flow cleanrooms or the impact of cooling fans
on cleanroom conditions.

Tracer particle ejection velocity

Most aerosol generators, ultrasonic nebulizers and fog machines
either by heat, pressurization or a fan, propel the tracer particles
out of the generator and into a dispersion manifold. The velocity of
the tracer particles leaving the machine or manifold is the ejection
velocity. The ejection velocity alone may misrepresent the actual air
flow within the cleanroom if the ejection velocity is so great that
tracer particles are jetted into the air stream.

The effects of large particles and ejection velocity

Figure 17 (a) and (b) demonstrate the effects of ejection velocity
relationship to tracer particles size and show the effects of large
particles and a high ejection velocity.

Even without airflow, the ejection velocity combined with the

larger particle size, the tracer particles indicate air flow, moving in a
mostly downward direction (Figure 17(b)).

Figure 18 (a) and (b) shows the effects of small particles and a high
ejection rate. Figure 18(b) shows the high ejection rate of the tracer
particles exiting the manifold, the energy is dissipated quickly, after
15 cm of travel, the smaller particles disperse into the environment
and are buoyant, providing a clear indication that there is no airflow
in this cleanroom.
192 Cleanroom Contamination Prevention and Control
Figure 17
(a) Ultrasonic fogger output (b) Ultrasonic fogger output
with unidirectional airflow without airflow

Photos taken by Microrite

Figure 18
(a) Output of theatrical smoke (b) Output of theatrical smoke
machine with unidirectional without airflow
airflow

Photos taken by Microrite

However, regardless of the type of method or media used in

performing a smoke study, the cleanroom must be cleaned as
thoroughly as after a facility shut down. The reason for this is
the type and variety of equipment, such as the smoke generator,
manifolds, tubing, cables stands, cameras, tripods and additional
 Air Flow Visualization 193

personnel involved in performing an AFV/smoke study as well as

the AFV/smoke media itself.

CONCLUSION
Air flow visualization studies are a required test for the qualification
of pharmaceutical cleanrooms and an extremely useful tool for
contamination control. Frequent FDA 483 observations as well as
warning letters highlight that this test is not well understood by end
users as well as equipment suppliers and cleanroom builders and
testing agencies.

Improperly conducted air flow visualization studies can lead

to incorrect conclusions related to the suitability of cleanrooms
and barrier systems for aseptic or low bioburden operations.
These mistakes can show up in failed media fills, hard to explain
environmental monitoring excursions, sterility test failures,
contaminated product and patient harm.

Properly conducted air flow visualization studies can be used

to characterize air flows patterns and identify areas where the
contamination control effect of clean air may need to be modified
or addressed as part of a risk based environmental monitoring
program.

REFERENCES
Avallone, H. (1989) Current regulatory issues regarding parenteral
inspections. Journal of Parenteral Science and Technology 43(1): 3–7.

CETA (2006) CETA Certification Guide for Sterile Compounding

Facilities CAG-003-2006.

EU GMP (2008) Annex 1: Manufacture of Sterile Medicinal Products

– revision November, 2008.
194 Cleanroom Contamination Prevention and Control

Food and Drug Administration (FDA) (2004) FDA Guidance for

Industry Sterile Drug Products Produced by Aseptic Processing
– Current Good Manufacturing Practice.

Hinds, W.C. (1999) Standard density spheres at 20° C and 1 atm. Aerosol
Technology. Wiley-Interscience.

International Organization for Standardization (ISO) (2015a) ISO

14644-1:2015 Cleanrooms and associated controlled environ-
ments Part 1: Classification of air cleanliness by particle
concentration.

International Organization for Standardization (ISO) (2015b)

ISO 14644-2:2015 Cleanrooms and associated controlled
environments — Part 2: Monitoring to provide evidence of
cleanroom performance related to air cleanliness by particle
concentration.

International Organization for Standardization (ISO) (2005) ISO

14644-3:2005 Cleanrooms and associated controlled environ-
ments Part 3: Test methods.

Ljungqvist, B., Reinmüller. B. (1996) Clean Room Design: Minimizing

Contamination Through Proper Design. AbeBooks. CRC Press.

Ljungqvist, B., Reinmüller, B. (1993) Interaction between air

movements and the dispersion of contaminants: clean zones
with unidirectional air flow. Journal of Parenteral Science and
Technology 47(2): 60–69.

PIC/S GMP Guide (PE 009-11) Annex 1 Clause 3, 53, 54.

USP <1116>, Microbiological Control and Monitoring of Aseptic

Processing Environments
 Air Flow Visualization 195

USP <797> Pharmaceutical Compounding Sterile Preparations.

WHO GMP for Sterile Pharmaceutical Products Working Document

Clause.

ABOUT THE AUTHOR

Morgan Polen is a subject matter expert in the field of contamination
control, airflow visualization and particle monitoring in cleanrooms.
Morgan has over 35 years of industry experience working in
cleanrooms in all industries.

Morgan’s Technical Leadership:

• Member of International Disk Drive and Equipment Materials
Association (IDEMA) Standards Committee.
• Member of US Technical Advisory Group to ISO Technical
Committee 209 (international cleanroom standards i.e., ISO
14644 Cleanrooms and Associated Controlled Environments).
• Member of ASTM Subcommittee E55.06 Working Group “Guide
for Critical Airflow Visualization”.
• Board member of the Institute of Environmental Sciences and
Technology (IEST).

Morgan has been instrumental in drafting and editing international

cleanroom standards, contamination control guidelines and best
practices. He has extensive experience working on cleanroom
projects in the United States, Canada, Mexico, Germany, Malaysia,
Taiwan, South Korea, Singapore, Thailand, United Kingdom, Ireland,
China, Philippines, India and Turkey and is a valuable resource in
addressing contamination control in critical environments for the
electronics, aerospace and healthcare industries.
196 Cleanroom Contamination Prevention and Control

As a key member of Microrite’s Expert Contamination

Control team, Morgan is instrumental in development of proactive
contamination control strategies through pragmatic risk assessment,
particle testing and airflow visualization. Morgan’s broad experience
troubleshooting contamination issues has helped companies with
FDA 483/warning letter remediation.
 7

STATIC CHARGE:
THE UNSEEN CONTAMINANT

Larry Levit
LBL Scientific
Alamo, CA
USA

Most people associate static electricity with damage to micro-

electronics (transistors, integrated circuits and hard disk drives)
from the little blue sparks resulting from static discharge. While
this is indeed a consequence of static charge buildup, it is not
considered a major concern in manufacturing pharmaceuticals
or medical devices. To make matters worse, traditionally, a static
control program is not implemented in most healthcare device
manufacturing facilities because it is not well recognized as a direct
or indirect source of contamination. This unseen contaminant is
worth understanding and exploring in healthcare manufacturing.

UNDESIRABLE EFFECTS OF STATIC ELECTRICITY

Electrostatic forces
The greatest electrostatic concern in pharmaceutical and medical
device manufacturing is the electrostatic force between objects in the
manufacturing process. This force is called an “action at a distance”

197
198 Cleanroom Contamination Prevention and Control

because objects which are charged even when not in direct contact
can experience a force on them. Two charged objects will repel each
other if they have the same charge polarity. If they have opposite
polarities, they will attract (Figure 1).

Figure 1 Electrostatic forces on adjacent objects which need not

be in physical contact

Illustration by Dr. Levit

These forces can be surprisingly large. In normal cleanroom opera-

tion, aerosol particles are consistently drawn off the unidirectional
flow of the cleanroom and are pulled onto the surface of the object
intended to be kept clean. Unfortunately, static attraction cancels
out the cleansing effect of the cleanroom (Figure 2).

Figure 2 Static attraction canceling out the cleansing effect

of the cleanroom

Illustration by Dr. Levit

 Static Charge: The Unseen Contaminant 199

The rate of electrostatic attraction (ESA) of small particles to charged

objects has been calculated (Donovan, 1990; Cooper et al., 1988), and
particle deposition data recorded in cleanrooms match the calculation
(Levit et al., 2000). The importance of ESA becomes greater as the
particle size becomes smaller, and below 100 nm is the dominant
effect in contamination levels as compared to sedimentation
(gravity), aerodynamics (wind) and diffusion (Brownian motion).
This effect makes particles that land on charged product while being
processed in the cleanroom stick to the surface of the product so
tenaciously that blowing off with compressed air will not dislodge
them. Objects like IV bags or plastic catheters fall victim to static-
driven micro-contamination.

Figure 3 Micro photograph of a glass disk in manufacturing

NO STATIC CHARGE CONTROL WITH STATIC CHARGE CONTROL

Photographs taken by Dr. Levit

To illustrate the effects of static attraction, a process in the

manufacturing of glass disks had static control (ionizers) added.
Sample microphotographs before and after the static control was
added is shown in Figure 3.

Other cases in which electrostatic forces can be significant

include both attraction and repulsive forces. For example, visible
contamination on the inside of an IV bag that cannot be brushed or
blown away due to the static charge on the bag as the contamination
is trapped within during the manufacturing process. Powders are,
in general, insulators and as such, charge up easily. It is common to
200 Cleanroom Contamination Prevention and Control

see powders sticking to glassware so strongly that they cannot be

blown off with compressed air. In other examples, pills jumping out
of a waffle pack in automated packaging is a common phenomenon.
Another repulsive force that has been observed involves pouring
powder into a container and having it fly up into the air rather than
settling into the container. Products which are insulators themselves,
like Teflon™ or polyamide, are a serious contamination issue. Small
particles stick to the charged product and strongly resist efforts to
remove them.

Interference with robotics

When a charged object approaches a conductor at low voltage
or grounded, the charge will jump for the object to the adjacent
conductor. In metal-to-metal discharges, the discharge is extremely
rapid (< 1 ns.). Such a discharge is an efficient radiator of high
frequency (microwave) radio waves. These waves can bounce off
objects in the environment, penetrate through tight spaces and
induce high speed voltage pulses on conductors within. One such
discharge is shown in Figure 4.

Figure 4 The pulse is extremely short and shows reflections off

objects at 30 cm and at 45 cm

Image of oscilloscope output taken by Dr. Levit

 Static Charge: The Unseen Contaminant 201

These pulses are capable of corrupting data or program instructions

(digital words) within the microcontroller which drives the robot.
Such a pulse must occur just as the digital word is latched into the
microcontroller. If the discharge is a few nanoseconds early or late,
no issue is developed but correctly timed, it can be disastrous.

One step in an automated line can stop operating, causing

the line to shut down or create a “traffic jam.” Equally likely, the
process step can fail to perform the right action or make an incorrect
measurement. For example, a robot can send components to the
wrong location, causing havoc to the line. Either of these looks very
much like an intermittent software bug which results in numerous
service calls to the vendor responsible for the equipment but no
bug is found. The result is significant down time for the line and
lower output of functioning parts. Because of the statistical nature
of the timing, such effects happen only occasionally and at random.
This sort of effect might occur once a day or once a week, making it
extremely hard to diagnose and fix.

Electrostatic discharge damage

While most medical devices and pharmaceuticals are rarely sensitive
to such discharges, in some cases they are. Any such product
which involves electronics is susceptible to physical damage due
to electrostatic discharge (ESD). Cardiac pacemakers are a good
example. In particular, damage caused by charge on a circuit board
is commonly the driver for electrostatic discharge damage.

Static discharge deposits energy in a small volume and if the

discharge is from a metal object to another metal object, as discussed
above, it is extremely rapid. When the energy in the discharge exceeds
a threshold level, flammable vapor or powder can be ignited and cause
an explosion. The threshold is called the minimum ignition energy
and is tabulated in a variety of places (Euratex, 2020). The discharge
requires both a flammable vapor and oxygen. The ratio of vapor to
oxygen can vary but for a stoichiometric mixture (exactly the correct
202 Cleanroom Contamination Prevention and Control

amount of oxygen for the amount of vapor present), the least amount
of energy is required for an explosion.

UNDERLYING SCIENCE
Static charge generation
Whenever two dissimilar surfaces placed in close contact are
separated, one surface loses electrons and becomes positively charged,
while the other surface gains electrons and becomes negatively
charged. This is known as triboelectric charging. Any material – solid
or liquid, may be charged triboelectrically by friction, separation of
materials, or fluid flow however, gasses cannot charge in this way.
The contaminating particles in the gas can, however, cause slow tribo
charging to occur. The magnitude of the charge will be affected by
the surface condition, area of contact, speed of separation or rubbing,
and the humidity. Whether the material remains charged depends
on its conductivity and the availability of a path for the charge to flow
to ground. If charge is allowed to accumulate on a material it may
attract and bond particles to its surface.

The principle of triboelectric charging is related to the work

function of each material. This is defined as the energy required to
remove an electron from the surface of the material. Since the work
function is related to the electronic energy levels of the material,
the work function of each material is unique. Thus, any time
two materials are placed in contact with each other, electrons are
transferred from one material to the other. The amount of transfer
is dependent upon the nature of the surfaces and the nature of
the contact. Smooth surfaces with a significant amount of contact
pressure and relative motion (sliding) result in a greater amount of
charge transfer.

When the materials are separated, the capacitance between

them decreases so the potential difference (voltage) between them
increases. Thus, the mechanism of triboelectric charging results from
contact of dissimilar materials and separation of them afterward.
 Static Charge: The Unseen Contaminant 203

Induction
Static charge is also generated by induction. Static charge on an
object can create or “induce” opposite polarity charges on the sur­
face of another object by causing the positive and negative charges
on the object to separate. As with charge generated triboelectrically,
the induced charges can attract particles and contact with ground
can result in damaging ESD events and electromagnetic inter­
ference (EMI).

The cleanroom environment

The environment of a cleanroom lends itself to increased levels
of tribocharging and decreased dissipation of the charge from
the surface of insulators as compared with levels on insulators in
a conventional room. First, any contamination that exists on the
surface of an insulator provides a discharge path to ground for
the charge. In a cleanroom, where there is concern about attracting
particles to surfaces, all objects can be wiped down to eliminate this
discharge mechanism.

Studies (Levit and Guan, 2001) have shown the relationship

between humidity and tribocharging. In a location with 60%
relative humidity, an equilibrium is achieved between water vapor
and the surface of each material. At a lower humidity (~35–45%),
the equilibrium condition dictates that less water vapor will be
present in the surface of the material. This water vapor modulates
the “work function” of the material, placing its value part way
between the work function of the material and the work function of
water. Thus, materials in a low humidity environment exhibit more
triboelectric charging than those is a higher humidity environment.
Since cleanrooms are maintained at low humidity, more charging is
observed in the cleanroom than in the conventional room.
204 Cleanroom Contamination Prevention and Control

Insulators vs. conductors

All materials can be characterized as either electrical conductors or
as electrical insulators. A conductor is a material through which an
electrical current can pass and an insulator is a material through which
an electrical current cannot pass. There is a good deal of variation
in the electrical properties of conductors (electrical resistivity) that
allows electrical conductors to be further classified as conductors
(good conductors and dissipative or poor conductors) but both are
differentiated from insulators by their ability to sustain an electrical
current. The parameter of electrical resistivity is a measure of the
voltage that must be applied to a sample of the material to stand a
unit current through a cubic sample of the material.

Conductive materials (ESD, 1994) are defined as those having

a surface resistivity less than 1 × 104 Ώ cm. With a low electrical
resistance, electrons flow easily across the surface or through the bulk
of these materials. Charges go to ground or to another conductive
object that the material contacts. Metals like copper and aluminum
have exceptional low resistivities on the order of 10-8 Ώ cm, making
them extremely good conductors of electricity.

Dissipative materials (ANSI/ESD S541-2019) have a resistivity

equal to or greater than 1 × 104 Ώ cm but less than 1 × 1011 Ώ cm. For
these materials, the charges flow to ground more slowly and in a
somewhat more controlled manner than with conductive materials.
Dissipative materials are used for ESD control because they discharge
so slow that they often avoid ESD damage to microstructures that
are manufactured in high technology cleanrooms.

Insulative materials (Kohani and Pecht, 2017) are defined as those

materials with a resistivity greater than 1 × 1011 Ώ/sq. Because of the
incredible variation (>1020) in the resistivity of materials, it is clear
that the ability of an insulator to dissipate surface charge is so poor
that it can be ignored. For a conductor, this corresponds to a time of
much less than a nanosecond. For a fairly good insulator (>1013 Ώ/
sq), this corresponds to >1000 seconds. For extremely good insulators
like quartz, Teflon, the material can be assumed to never discharge.
 Static Charge: The Unseen Contaminant 205

Thus, in a cleanroom where high levels of charge are developed on

such surfaces, they will build up over the period of days, achieving
extremely high levels. Grounding one of these extremely good insulat-
ors accomplishes nothing and the charge remains on the surface.

Using conductors and dissipative materials

to shape a discharge
A discharge between two metal objects occurs virtually unimpeded.
There is only a tiny fraction of an Ohm in the discharge path. In
comparison, if at least one of the two objects is made of dissipative
material or has a dissipative coating, the peak current in the discharge
and the time for a discharge is dramatically increased. The difference
in the magnitude of the discharge currents is dramatic.

Figure 5 Electrical discharges to a metal object (black) and

through a series resistance of ESD safe tools (grey)

Graph generated by Dr. Levit

Figure 5 above illustrates this difference for a relatively low series

resistance (10 Ώ) and a much higher one (1000 Ώ). The peak current
sets the maximum instantaneous power in the discharge and the tail
of the pulse determines what the peak temperature will be at the
point of discharge. Both favor the high resistance for the protection
of the product. Use of dissipative tools and robotic elements are
commonly used to generate an ESD safe environment with respect
to discharge.
206 Cleanroom Contamination Prevention and Control

CLEANROOM ELECTROSTATIC MANAGEMENT

Required insulators
An arsenal of methods is employed to deal with static charge. The
fundamental rule and the first step in an electrostatic management
program is the elimination of insulators wherever possible. As
discussed above, insulators hold their static charge indefinitely and
the act of grounding them accomplishes nothing. Common insul­
ators include operators’ gloves, plastic walls separating automated
steps in a process, plastic parts bins and plastic sheet protectors
protecting paper documents as well as glassware used for holding
liquids and chemicals. There are static dissipative versions for most
of these items which should be considered (Figure 6).

Figure 6(A)Electrostatic discharge 6(B) Electrostatic discharge

safe tweezers hazard tweezers

Image courtesy of TDI, International, Inc.

Unfortunately, the product itself may be an insulator and cannot

be eliminated. In that case other steps must be taken including
triboelectric matching of the product to the item handling it (i.e.,
gloves) and the use of air ionization, described below.
 Static Charge: The Unseen Contaminant 207

Grounding
The defense for static charge on conductors is to ground them. This
draws the static charge off the conductor and sends it to ground
leaving the conductor neutral. The usual mitigation techniques are
grounding of all facilities components (walls, floors, workbenches,
and equipment), appropriate use of conductive and static dissipative
materials, and local or ceiling mounted ionization to control static
charge on insulators. Education of cleanroom personnel in the
practice of static control, as well as auditing is extremely important.

Modern cleanroom environments make extensive use of

grounding with conductive and static dissipative materials as a
component of a program to control electrostatic charge. Grounding
prevents the generation of static charge on materials that are
connected to ground. If a conductive or static dissipative material
does become charged, connecting it to ground will remove this
stored charge. To be effective in controlling static charge, conductive
and static dissipative materials must be provided with a reliable path
for the static charge to flow to ground. The success of any grounding
method depends on the integrity of the ground path. In critical
applications, ground path monitoring and periodic verification are
used as part of a static control program.

By creating the ground path, the static charge on equipment,

materials, and personnel, can be rapidly, and harmlessly,
neutralized. Ground connections should exist for most accessible
surfaces of production equipment. It is particularly important to
assure grounding of those surfaces within 300 mm (12 inches) of
static sensitive product. Care should also be taken with moving
parts of automated equipment. A ground connection that exists
when a robotic handler is stationary may be lost when the robot is
in motion. Flexible grounding connections or conductive lubricants
should be employed.

Static dissipative materials are used in the construction of

cleanrooms and mini-environments to reduce the accumulation of
static charge. Insulating walls or baffles should be avoided whenever
208 Cleanroom Contamination Prevention and Control

possible. Tweezers and dissipative work surfaces used in product

assembly are two examples of locations where dissipative materials
are the best material choice. In contrast with metal tweezers, metal
work surfaces, or insulative mini-environment walls, the cost of
the dissipative material is higher but often the payback in terms of
decreased amount of ESD damage makes the dissipative materials
a good investment. Static dissipative packaging materials are also
used to shield the product from static charge buildup or ESD damage
as it is transported between processes in the cleanroom.

Grounding methods and static dissipative materials are also

used to assure that charge does not accumulate or transfer from
personnel to sensitive products or equipment. A variety of personnel
grounding methods are employed using garments, booties, gloves,
static dissipative flooring, ESD chairs and wrist or heel straps (Figure
7). For successful static charge control, all of these methods must be
monitored and verified periodically.

Figure 7 Special footwear to provide electrical conductivity

to operators on grounded ESD floors or ESD mats

Courtesy of Transforming Technologies

 Static Charge: The Unseen Contaminant 209

For grounding of chairs or personnel through footwear, the floor

must have electrical conductivity to ground. This means a specially
designed floor (called an ESD floor) is required. If no such floor is
installed, a second choice is to use grounded static dissipative mats
at each work station (Figure 8).

Figure 8 Utilizing grounded mats in a manufacturing cleanroom

Courtesy of Static Control Solutions (SCS)

One of the important sources of electric fields is the garments and

undergarments worn by workers. To deal with this issue, garments
are available with electrical conductivity across the garment. The
cloth used contains a matrix of electrically conducting carbon
threads. This provides electrical conductivity across the garment
and serves as a Faraday shield against fields from garments or
undergarments (Figure 9).

The simplest field suppression system consists of smocks made

of appropriate material but they are also available with a ground
snap which, when used, provides much greater field suppression.

In more stringent cleanroom applications, an entire bunny suit

(coverall) with ESD threads is used. Care is taken to assure electrical
conductivity between the hood, the body and the boots.
210 Cleanroom Contamination Prevention and Control
Figure 9 ESD garments utilize special fabrics to achieve electrical
conductivity of the garment material

Courtesy of Transforming Technologies

For field sensitive applications where fields can be a significant driver

of micro contamination, the garment has a body contact point (BCP)
which often is comprised of conducting fabric cuffs which contact
the skin of the operator. Such a configuration is recommended for
manual operations involving plastic components as is the case for
medical device manufacturing.

One very useful static control system is the use of garments

with a BCP and either ESD shoes or heel grounders (see above) in
manufacturing cleanrooms where the floor has slight electrical
conductivity integrated into its design (called static dissipative).
The special so-called ESD floor must be electrically grounded so it
provides a path to ground for carts and operators as they move about
the manufacturing floor.

Ionization
Unfortunately, not all insulators can be eliminated. For example,
glass or Teflon containers may be required for holding caustic
chemicals that would be attacked if they were made of dissipative
plastic. These insulators must be dealt with utilizing air ionization.
 Static Charge: The Unseen Contaminant 211

Air ionizers are devices that create a balanced population of

positive and negative air ions. These ions seek out surface static
charge and neutralize it. The positive ions discharge negative
charge and the negative ions discharge positive charge. These ions
are created by corona discharge (high voltage on needle points), by
x-rays or by alpha radioactivity. Care is taken in the design of these
ionizers to assure that the positive and negative ions are created in
equal numbers or the ionizer will charge the environment.

Unfortunately, the ions of both polarities find each other and

annihilate one another. Thus, the ionizer must be placed close to the
object to be neutralized. In some applications, the ions can be made
to move on unidirectional air flow to provide blanket protection of a
large area by placing the ionizers up to 3 m above the work surface.
These ionizers are called ceiling emitters. The performance of such
an ionizer gives only modest protection for a large area but fails
when there are objects obstructing the air flow. Such ionizers are an
excellent solution for ISO Class 4 or better cleanrooms but for Class 5
and above, fan driven ionization is highly preferred.

Metal obstructions will actually draw ions to them and eliminate

them by shunting their charge to ground. One case where this is a
serious issue is when production is executed under a microscope.
Ceiling emitters should not be used in production areas that utilize
work stations with personnel operating them.

For manual operations at work stations, blower-type ionizers

should be used. These can be mounted 0.5-1 m above each work-
station to provide significant ion flow to the work surface. For really
close-in work, such as under a microscope, single fan blowers should
be used within 50 cm or less of the operation.

SUMMARY
Cleanrooms are a perfect breeding ground for static charge
generation. The pristine surfaces and the low humidity along with
the preponderance of excellent insulators creates and holds the
212 Cleanroom Contamination Prevention and Control

charge. This charge drives contamination issues owing to static

attraction and, in cases where the product consists of very small
structures, physical damage, leading to yield loss can occur.

Static charge control is well known in the electronics industry

and used universally with excellent results. In the pharmaceutical
and healthcare product industries, these technologies are not at all
well known or even recognized. This is unfortunate because the
relatively modest investment in a static control program routinely
reaps substantial improvements in the profit of such production
lines.

The first step in a static control program is the elimination of as

many insulators as possible. They should be replaced by conducting
or static dissipative counterparts and they should be grounded.
Grounding is a critical part of a static control program. There are
a very large number of techniques used to provide ground. They
involve everything from grounded floors with electrical conductivity,
to special footwear to maintain ground for operators. A simple wire
from ground to a fixture works well and making sure the “skin”
of a process tool is grounded is also required. Figure 10 below lists
certain recalls which highlight the importance of static control.

Attention to such detail will help an operation run smoothly and

maximize yield.
 Static Charge: The Unseen Contaminant 213
Figure 10 Malfunctions of medical devices due to electrostatic
occurrences: Big data analysis of 10 years of the FDA’s Reports

Number
Number Number of non-
Apparent causes and/or failure
Device type of ESD of deaths/ recoverable/
modes of ESD events
malfunctions injuries recoverable
malfunctions

Clinical 769 0/12 5/764 Buildup of static charge on the

chemistry sample holder or the plastic
analyzer diffuser (due to low RH); user
touched the metallic barcode; ESD
resulted in inaccurate readings or
error messages on the screen
Infusion pump 173 0/6 167/6 Lightning or touching keypad or
screen while battery replacement;
ESD caused date and time changes
in the log files, unexpected restart,
or wrong dose delivery
Heart assist 122 4/4 89/33 The surgeon touched the device
devices with surgical tools; the patient
(wearable was wearing a silk suit; riding in a
defibrillators, car; sitting on a sofa; plugging in a
ventricular laptop; or replacing batteries; ESD
assist devices) resulted temporary pump motor
controller restart
Neuro- 33 1/3 17/16 Patient touched the programmer,
stimulator while wearing a static dissipation
belt on waist that caused shocking
sensation, loss of therapy,
unexpected rest; ESD resulted in
wrong values for remaining battery
life
Imaging 19 0/0 2/17 While the mobile system was
system being moved down a ramp;
(portable touched a metal frame or
X-ray, MRI, entered an elevator; ESD caused
CT) unintended sporadic movement of
wheels, and artifacts displayed on
the screen
Cochlear 18 0/4 18/0 During surgery, the surgeon
implant touched the hex screw of the
device, while patient was sliding
down a plastic slide
214 Cleanroom Contamination Prevention and Control

Number
Number Number of non-
Apparent causes and/or failure
Device type of ESD of deaths/ recoverable/
modes of ESD events
malfunctions injuries recoverable
malfunctions

Pulse 18 0/0 0/18 When the surgeon touched the

generator hex screw of the device, ESD
resulted in wrong values and error
messages for remaining battery life
Ventilator 17 0/2 8/9 A charged user touched the
operation panel with hand or
stethoscope during intra-hospital
patient transport; while changing
the bed sheet and connecting
an ungrounded cable to a
port; ESD caused intermittent
communication, unexpected
shutdown, and inaccurate values
Pain relief 13 0/0 0/13 Static charge from lightning caused
stimulator shocking sensation
Anesthesia 10 0/0 0/10 When a charged user touched
unit a metallic arm of the device,
ESD caused intermittent
communication, unexpected
shutdown, or inaccurate values
Heart/lung 8 0/0 0/8 Static charge created due to
machine friction of the roller pump with
PVC tubes caused noise in the
ECG data (probably due to low
RH)
Blood glucose 9 0/2 0 /9 Time and date was changed due
monitor to ESD
Battery 6 0/0 0/6 ESD caused communication error
charger and displayed a “replace battery”
message
Intracranial 4 0/4 0/4 Inaccurate reading after ESD
pressure events
monitor
Eye surgery 4 0/3 0/4 ESD caused shock to users
device
Surgery 3 0/0 0/3 Triboelectric charging of the
instrument sensor with disposable plastics
caused intermittently delayed
response
 Static Charge: The Unseen Contaminant 215

Number
Number Number of non-
Apparent causes and/or failure
Device type of ESD of deaths/ recoverable/
modes of ESD events
malfunctions injuries recoverable
malfunctions

Cardio- 2 2/0 0/2 Static charge build up from the

pulmonary handpiece caused ESD shocks to
bypass users
monitor
Dermatology 2 0/0 0/1 ESD caused inaccurate reading,
laser which was reproduced by
exceeding ESD test criteria
Slit lamp 2 0/2 2/0 ESD caused inaccurate reading,
which was reproduced by
exceeding ESD test criteria
Powered 1 0/0 0/1 ESD caused temporary lockup of
patient bed motor controller board
Cell counter 2 0/0 0/2 ESD caused unexpected shut
down and shock to user while
plugging cables into a USB
connector
Pulse 1 0/0 0/2 ESD testing on the USB port and
oximeter chassis caused temporarily frozen
screen
Powered 1 0/0 1 /0 Triboelectric charging of the
wheelchair joystick caused ESD malfunction
1 0/0 0/1 ESD caused unexpected reset
after the patient (wearing a wool
sweater) touched a metal bar
Dentistry 1 0/0 1/0 Static charges accumulated on the
machine doctor’s hand possibly caused ESD
shocks to users
Patient lift 1 0/0 1/0 ES D malfunction occurred when
the nurse pressed a button of the
device
Laparoscope 1 0/0 0/1 Buildup of static charge on the
camera keypad due to poor grounding
of the camera head caused ESD
malfunction
Wound 1 0/0 1/0 ESD malfunction occurred when
therapy pump a nurse (wearing a nylon gown)
touched a button of the device
Coagulation 1 0/0 1/0 Static charge buildup due to low
analyzer RH caused ESD malfunction
216 Cleanroom Contamination Prevention and Control

Number
Number Number of non-
Apparent causes and/or failure
Device type of ESD of deaths/ recoverable/
modes of ESD events
malfunctions injuries recoverable
malfunctions

Tissue 1 0/0 1/0 ESD malfunction occurred when a

processor charged user touched the device’s
lid when RH was too low
Nebulizer 1 0/0 1/0 ESD malfunction occurred while
plugging the device into an adapter
Aspiration 1 0/0 1/0 –
pump

Source: Kohani and Pecht (2017)

REFERENCES
ANSI/ESD S541-2019 https://webstore.ansi.org/Standards/ESDA/ANSI
ESDS5412019?gclid=Cj0KCQjwuJz3BRDTARIsAMg-HxW-WzsCt
fM6cZC5F8gMALdVEIJips5FYCFPs9l0pZRPPHE93OKzCRUaAq
xbEALw_wcB ANSI Webstore. Accessed June 16, 2020.

Cooper, D.W., Miller, R.J., Wu, J.J., Peters, M.H. (1988) Deposition
of Submicron Aerosol Particles During Integrated Circuit
Manufacturing: Theory. Particulate Science and Technology 8(3–4):
27–32.

Donavan, R.P. (1990) Particle Control for Semiconductor Manufacturing.

Marcel Dekker, New York.

ESD (1994) ESD Association Advisory for Protection and Sensitivity

Testing of Electrostatic Discharge Susceptible Items – Handbook.
Rome, NY: Electrostatic Discharge Association.

Euratex, May 5, 2020. “Dust Classification.” http://www.euratex.co.uk/

dust-classification/
 Static Charge: The Unseen Contaminant 217

Kohani, M., Pecht, M. (2017) “Malfunctions of Medical Devices Due

to Electrostatic Occurrences Big Data Analysis of 10 Years of the
FDA’s Reports.” IEEE Access 6 (December 11, 2017): 5805–11.

Levit, L.B., Hanley, T.M., Curran, F. (2000) In 300 mm Contamination

Control, Watch out for Electrostatic Attraction. Solid State
Technology 43(6): 209–214.

Levit, L.B., Guan, W. (2001) Measuring Tribocharging Efficiency in

Varying Atmospheric Humidity and Nitrogen. Proceedings of
the Annual Conference of the Electrostatics Society of America,
June 27–30, 2001.

ABOUT THE AUTHOR

Dr. Lawrence B. Levit graduated with honors from Case Institute of
Technology in 1964 and received a Ph.D. from Case Western Reserve
University in 1970. He is a member of the American Physical Society,
the Electrostatics Society of America, the Institute for Environmental
Sciences Technology and the Electrostatic Discharge Association. At
IEST, he chairs the Working Group RP-CC-022 on electrostatics in the
cleanroom. He is a vetted instructor for the Electrostatic Discharge
Association and the IEST on the subjects of Digital Oscilloscope
operation, static charge control and Cleanroom Technology. He is
a frequent lecturer on electrostatic charge control techniques in the
United States, Europe and Asia. Dr. Levit was a faculty member at
Louisiana State University (physics) and worked 20 years at LeCroy
Research Systems as Chief Scientist. LeCroy provided high speed
electronics to the High Energy Physics research community. He
helped instrument five Nobel Prize winning experiments. His career
also included 20 years at Ion Systems in Northern California as Chief
Scientist where he developed static electricity control techniques
for the semiconductor industry worldwide. More recently, he has
focused on electrostatics in pharmaceutical and medical device
manufacturing including a partnership with Microrite, Inc.
 8

DESIGNING, COMMISSIONING AND

RISK ASSESSING CLEANROOMS FOR
BIOCONTAMINATION CONTROL

Tim Sandle
PharmaceuticalMicrobiology
Interest Group (Pharmig)
UK

INTRODUCTION
Cleanrooms are highly controlled environments where the air qual­
ity needs to be designed ensure that the high standards of cleanliness
required for the manufacture of pharmaceutical and healthcare
products are met. In addition, cleanrooms need to be designed
so they easily cleaned, disinfected and maintained. Together will
appropriately gowned and trained personnel (Ljungqvist and
Reinmüller, 2003), cleanrooms play an essential part in achieving
biocontamination control (ISO 14698-1, 1998). This chapter is con­
cerned with the assessment of cleanrooms (whether this is called
qualification, validation or commissioning), which involves
consideration of cleanroom design parameters together with the
testing necessary to demonstrate that the design parameters are
being consistently met. With design, the minimization of microbial
contamination is the essential requirement

219
220 Cleanroom Contamination Prevention and Control

To achieve the stringent standards required for biocontamina-

tion control, high fresh air rates, extensive air filtering, temperature
and humidity control are each required. In addition, protection from
uncontrolled ingress of external ambient air is needed, and this is
achieved by creating a pressure differential between the cleanroom
and its surroundings. Through these measures, cleanrooms can
provide appropriately controlled environments for both sterile and
non-sterile pharmaceutical manufacturing.

This chapter examines the design and testing of cleanrooms and

clean air devices in the pharmaceutical and healthcare context. The
chapter briefly introduces cleanrooms and focuses on how these
specially constructed spaces are certified and classified, with a focus
upon microbiological control. In doing so, the chapter explains the
key aspects of the physical operation of cleanrooms, how these
need to be incorporated into a “quality by design” philosophy
and discusses on-going monitoring requirements, both “routine”
and for re-assessment purposes (which runs that quality cannot
be adequately assured by in-process and finished inspections and
testing but it should be built in to the manufacturing process)
(Sharma and Singh, 2013). In doing so, it is important to note there
are different regulatory expectations in terms of what is required
for cleanroom commissioning, particularly at the performance
qualification stage. This chapter provides general advice in relation
to achieving particle and microbial control; however, applicable
regulatory guidance must always be referred to.

The chapter also considers risks, and where, with due

consideration of risk, design and commissioning aspects can be
optimized. As an example, cleanrooms are highly energy intensive
to operate. Because the air volumes supplied to the cleanrooms are
many times (10–100) greater than those supplied to conventionally
ventilated rooms, the capital and operating costs for the construction
of cleanrooms can be very high. It can be the case that some cleanrooms
are over-specified and hence, with due consideration of the risks, it
is possible to review the level of cleanroom certification undertaken
and the way in which cleanrooms are designed and operated and
 Designing, Commissioning and Risk Assessing Cleanrooms 221

to make some adjustments while still maintain biocontamination

control. This chapter concludes with a consideration of where risk-
based cleanroom optimization can occur.

CLEANROOMS AND CLEAN AIR DEVICES

A cleanroom is a separated space where the level of airborne
particulates is controlled and where the indoor space is subject to set
environmental parameters. In pharmaceuticals and healthcare, the
operation of cleanrooms should form an essential part of a company’s
contamination control strategy. To be effective, the cleanroom must
be constructed and used in such a way as to minimize the generation
and retention of particles. The key aspect is that the level of cleanliness
is controlled. Achieving control is essential for each manufacturer’s
contamination control strategy, to avoid product contamination, to
protect the patient, and to meet global regulatory expectations.

While regulatory expectations have the ultimate focal point

of patient safety, there are some differences between regulators
(at least with terminology). A key difference between inspectorate
bodies is with cleanroom classification and categorization. For the
Food and Drug Administration (FDA) (2004) the ISO 14644 (Part
1, 2015) standard of cleanroom classification is used. ISO 14644 is
a general standard covering all industries that use cleanrooms
(from electronics to pharmaceuticals). A further difference arises in
Europe where a grading system, A to D, specified in the EU GMP
guide (2014), is used for normal cleanroom operation (although ISO
14644 is used for the validation). The World Health Organization
(WHO) (2011) uses the same grading system as Europe in its GMP
guidelines, as does the post-Brexit UK.

In terms of what is “clean” in the context of a cleanroom, particles

are the general expression of air cleanliness (or more precisely,
“particle concentration” within a given volume of air – usually a
cubic meter or cubic foot). “Particle” in the context of a cleanroom
is a general term for sub-visible matter. An airborne particles refers
to particles suspended in air. Air contains a variety of different
222 Cleanroom Contamination Prevention and Control

particles of a range of different sizes. These are particles of dust,

dirt, skin, microorganisms and so on. The function of a cleanroom is
to reduce the number of airborne particles to a level commensurate
to the cleanroom class. To put this into context, an office building air
contains from 500,000 to 1,000,000 particles (0.5 microns or larger)
per cubic foot of air. In contrast, an ISO Class 5/EU GMP Grade A
cleanroom is designed not to allow more than 100 particles (0.5
microns or larger) per cubic foot of air. A micron is a unit of length
equal to one millionth (10-6) of a meter.

An important sub-class of particles are viable particles (where

people are present a proportion of particles will be viable, although
the ratio is indeterminant) (Ljungqvist and Reinmüller, 2000). These
are particles consisting of, or most probably supporting, one or more
live microorganisms: single-cell organisms capable of multiplication
under favorable conditions (in the presence of water and nutrients
at an ambient temperature appropriate for multiplication). Such
microorganisms include bacteria, molds and yeasts. Typically, there
are free free-floating microorganisms and instead microorganisms
in cleanroom air are more likely to be found attached to rafts of
matter, such as fibers or skin cells (typically 33 µm × 44 µm, and
3 to 5 µm thick) (McIntosh et al., 1978) or fragments of skin cells
(which leads to the term “microbial carrying particle” sometimes
being used (Whyte, 1986)). While microbial carrying particles vary
in size, shape, and density, the average equivalent particle diameter
of microbial carrying particles in cleanroom air is about 12 µm
(Whyte and Hejab, 2007). Airborne microbial carrying particles in
ventilated rooms are generated within the room and most, if not
all, in a well-designed cleanroom, come from personnel. As well
as this form of airborne microorganisms, bacteria and fungi can be
found on surfaces (where particles settle out onto surfaces through
gravitational settling, turbulent deposition, Brownian diffusion, and
electrostatic attraction (Whyte et al., 2015); or via personnel touching
surfaces or by moving objects) and transient to personnel garments.

Cleanroom cleanliness is achieved through good design

principles (more specifically, the auspice of “Quality by Design”
 Designing, Commissioning and Risk Assessing Cleanrooms 223

and the principle of building-in quality at the outset) and verified

by classification (a point that is well developed by Friedman (2010),
in relation to cleanroom qualification). Once the classification is
established, a series of other environmental parameters must be
met. Cleanroom classification and the control and assessment of
these parameters are described later in this chapter. A classified
cleanroom is not necessarily the same as a controlled environment.
With a controlled environment, various user defined environmental
aspects are “controlled” (so that they conform to user expectations);
whereas, with a classified cleanroom, the cleanroom must conform
to an international standard (ordinarily ISO 14644). The controlled
environment must meet additional regulatory expectations and
guidance, such as those assessed through environmental monitoring.

Cleanrooms, as set out in ISO 14644 Part 1 (2015), have three

different “states” of use. These are:

• As built.
• At rest (sometimes referred to as “static state” or “unoccupied
state”).
• In operation (sometimes referred to as “dynamic state” or
“occupied state”).

With these terms, “as built” refers to the condition of a newly built
cleanroom, with the operational qualification having been completed,
at the point it is handed over to the user for performance qualification.
For “at rest” conditions, this is the room without personnel present,
following a “clean up time” or “recovery time” (which in EU GMP
is 15–20 minutes), and either with equipment operating normally or
with equipment not operating (there are differences in definition, and
it is important to define what “at rest” means to avoid confusion). “In
operation” is a less ambiguous term, and it is applied to cleanrooms
that are being used for normal processing activities with personnel
present and equipment operating.
224 Cleanroom Contamination Prevention and Control

The objective of a pharmaceutical facility cleanroom is to

maintain a clean air space and to avoid the product from becoming
contaminated from particles (including microorganisms) carried
in the air stream. There are many sources of contamination (Halls,
2004). For instance, the atmosphere contains dust, microorganisms,
condensates, and gases. Manufacturing processes will also produce
a range of contaminants. Wherever there is a process which grinds,
corrodes, fumes, heats, sprays, turns, and so on, particles and fumes
are emitted and will potentially contaminate the surroundings.

Furthermore, particles can be generated from a variety of

sources. These include (Sharp et al., 2010):

• Facilities, such as: walls, floors and ceilings; paint and coatings;
construction material; air conditioning debris; room air and
vapors; spills and leaks.
• People, including: skin flakes and oil; cosmetics and perfume;
spittle; clothing debris (lint, fibers etc.); hair.

• Equipment generated, including: friction and wear particles,

lubricants and emissions, vibrations.

• Cleaning equipment, like: brooms, mops and dusters; cleaning

chemicals.
• Fluids, arising from spillages.

• Particulates floating in air, primarily: bacteria, fungi, organic

material and moisture.

• Compressed gasses.

• Product generated.

However, it remains that people are the primary source of

contamination within the cleanroom space. Within cleanrooms the
primary concern is with those particles which are microorganisms
or likely to be carrying microorganisms, such as skin flakes
(Reinmüller, 2001). The major source, and hence the primary risk, is
from people. Most cleanroom microorganisms are suspended in the
 Designing, Commissioning and Risk Assessing Cleanrooms 225

air, originating from people. If they settle on a dry surface, they are
unlikely to survive. However, microorganisms can be transferred
by people touching surfaces. Such risks can be increased through
physical behavior like fast motion and horseplay or from physical
concerns like room temperature, humidity or from psychological
concerns like claustrophobia, odors and workplace attitude. In
general, people produce contamination via (Sandle, 2013a):

• Body regenerative processes: skin flakes, oils, perspiration and

hair.
• Behavior: rate of movement, sneezing and coughing.
• Attitude: work habits and communication between personnel.

Within cleanrooms various types of clean air devices may be found.

Sometimes these are referred to as “separative devices” (in order to
emphasize that there is some form of a barrier between the device
and the surrounding cleanroom). Clean air devices include clean air
hoods, gloveboxes, isolators and minienvironments. Such devices
normally operate at ISO 14644 Class 5 (approximately equivalent
to EU and WHO GMP Grade A). Related to these are biosafety or
microbiological safety cabinets, used for handling microorganisms
(either designed to protect the activity from external contamination
or to protect the operator from the material being handled, or,
sometimes both) (Sandle, 2013b).

Cleanrooms are generally designed to have turbulent airflow.

This is where the supply air entering the clean zone mixes with the
internal air by means of induction. With turbulent airflow, if the
mixing is perfect, the concentration of contamination in the clean
zone is the rate of generation of contamination divided by the
volume flow rate through the cleanroom.

Where most cleanrooms operate with a turbulent airflow, clean

air devices are most often designed to minimize turbulence (which
creates dust and dirt collection pockets by operating with the air
blowing in one direction – described as “unidirectional airflow”, or
226 Cleanroom Contamination Prevention and Control

what was once erroneously referred to as “laminar”). A more precise

definition of unidirectional airflow is: a controlled airflow through
the entire cross-section of a clean zone with a steady velocity and
approximately parallel airstreams. Here the design feature is to move
air away from the critical activity to ensure that any contamination
is blown away to a less critical area.

For some devices, gloves are fitted in order to restrict the number
of personnel interventions. Such devices are described as Restrictive
Access Barrier Systems (RABS). These stand partway between a
conventional cabinet and an isolator. There are two basic designs of
RABS, namely the passive RABS, where the central heating, ventilation
and air conditioning (HVAC) system of the facility provides the
required air quality in the aseptic core, and the active RABS, which
is a stand-alone unit equipped with its own air handling unit. This
provides the required conditions in the aseptic core, drawing its
supply air from the external background surrounding the filling line.
Isolators are superior to RABS because that the contamination risk
is reduced through the construction of a barrier between the critical
area (sometimes called the “micro-environment”) and the outside
environment (Agalloco et al., 2007). Furthermore, the entire inside
of the isolator can be decontaminated through a proven cycle using
a surface decontaminating agent (such as hydrogen peroxide). The
effectiveness of the decontamination cycle can be quantitatively
assessed (through the use of biological indicators) and reproduced, in
a way not possible within a cleanroom. Isolators are used for sterility
testing, aseptic filing and other applications where a very clean
environment is required (Zwischen, 2006).

Many cleanrooms contain pass-through hatches. These are

hatches with double doors that protect critical environments while
allowing transfer or materials to or from adjoining rooms. They are
typically installed within the walls of cleanrooms. The hatches allow
materials to be transferred with minimal loss of room pressure and
without the need for personnel movement between rooms.
 Designing, Commissioning and Risk Assessing Cleanrooms 227

Another feature of cleanrooms are airlocks. An airlock is an

airtight room which adjoins two cleanrooms. The airlock acts as a
buffer zone between two independent areas of unequal pressure. A
pressure differential of ≥15 Pascals is typically maintained between
the inner room and the air lock; and between the air lock and the
external area.

CLEANROOMS DESIGN
Cleanrooms need to be designed in such a way that the objective for
contamination control can be achieved, and this should be central
to the design concept (and in tune with the “quality by design”
philosophy espoused by regulators – an approach that aims to
ensure the quality of medicines by employing statistical, analytical
and risk-management methodology in the design of any aspect of
pharmaceutical manufacture, be that a process, product, equipment
or a cleanroom) (ICH, 2009). Once designed, the cleanroom can
be commissioned. To design the cleanroom an understanding of
contamination sources is required (as discussed above) and the
fundamentals of air as a contamination control measure. There are
four principles applying to control of air-borne microorganisms in
cleanrooms. These are (Sandle, 2013c):

• Filtration (through the use of air filters). The air entering a

cleanroom from outside is filtered to exclude dust, and the
air inside is constantly re-circulated through High Efficiency
Particulate Air (HEPA) filters (or less commonly, ULPA filters –
Ultra Low Particulate Air (filter)). These are operated as part of
the overall HVAC system.

• Dilution, to ensure that particles generated in cleanrooms, in

addition to those which pass through the filters, are reduced
in concentration before carried out of the cleanroom via room
extracts. This is achieved diluting the clean air space with
incoming new “clean” air.
228 Cleanroom Contamination Prevention and Control

• Directional air flow, to ensure that air blows away from

critical zones, as particles and microorganisms cannot “swim
upstream” against a directional air flow. For this, some
cleanrooms are kept at a higher air pressure so that if there are
any leaks, air leaks out of the chamber instead of unfiltered air
coming in. Hence the static pressure between cleanrooms of
different class, and cleanrooms and unclassified areas can be
established and maintained using various airflow balancing
techniques. These include both active/automated and passive/
manual systems. Pressurization is defined as a technique that air
pressure differences are created mechanically between rooms
to introduce intentional air movement paths through room
leakage openings. With this the relative quantities of air that
are delivered and removed from each space by the ducted air
system, air transfer system and losses. These openings could be
either designated, such as doorways, or undesignated, such as
air gaps around doorframes or other cracks.

• Air movement. Rapid air movement within the cleanroom

is important for as long as particles and microorganisms stay
suspended in the air they are less of a problem, for when they
settle out onto a surface that they become a more critical cause
of contamination.

These principles are key to cleanroom design.

The starting point for cleanroom design is the ISO standards

that fall within the 14644 series. In reviewing these standards, it
is important to note that these standards are multi-industry and
they have not been specifically designed for pharmaceuticals and
healthcare. From this, the standards will contain some tests that are
unnecessary (such as assessing nano-particles); some tests that are
irrelevant (chemical bonding on surfaces); and there will be aspects
that are not covered. Most notably, an assessment of microbial
levels does not form part of the ISO series and here regulatory
guidance and suitable standards need to be referred to (such as
ISO 14698, EN 17141, USP <1116> and so forth). One common error
 Designing, Commissioning and Risk Assessing Cleanrooms 229

within the industry is where ISO 14644 methodology is used for

environmental monitoring sample site selection. This is the wrong
approach. The ISO approach to sampling is for the classification
of airborne particles only. It is not intended for particle count
monitoring outside of classification and it is not suitable for viable
monitoring. Sample locations should be based on a documented
risk assessment (applying a process flow methodology such as
Hazard Analysis and Critical Control Points (HACCP)). Microbial
assessment as part of cleanroom commissioning is addressed later
in this chapter.

This leads to a number of physical parameters which need

to be incorporated into the design. To design a cleanroom with
contamination control in mind; key factors must be accounted
for. These areas which combine engineering logistics and a bio­
contamination control perspective include (Sandle, 2015):

• Assigning the correct cleanliness level.

• Having optimal air change rates.
• Considering use of mini-environments (such as isolators, where
appropriate).
• Optimizing ceiling coverage.
• Develop cleanroom protocols.
• Assigning pressure differentials between cleanrooms of different
grades and minimizing pressure drops (air flow resistance).
• Location of large air handlers, ideally positioned close to point
of use.
• Having adequate sizing and minimize length of ductwork.
• Provide adequate space for low pressure drop air flow.
• Low face velocity.
• Use of variable speed fans.
• Optimizing pressurization.
230 Cleanroom Contamination Prevention and Control

• Consider air flow reduction when unoccupied.

• Motor efficiency.
• Defining HEPA filters differential pressures (expressed as ΔP).
• Determining electrical systems that power air systems.

Computational fluid dynamics (CFD) (the examination of fluid

flow in accordance with its physical properties such as velocity,
pressure, temperature, density and viscosity) is a useful tool for
predicting airflows or for finding solutions where the airflows are
not removing contamination as anticipated (Kauffman et al., 2016).
The use of CFD must always be supported by the use of airflow
visualization. This is because the CFD models are theoretical
abstracts (albeit based on data inputs), yet the visualization of air
through a well-designed “smoke” study provides direct evidence of
satisfactory (or unsatisfactory) air movement. In addition, airflow
visualization can be helpful when considering sample site selection
as part of the environmental monitoring program.

Other than air, other measures are required to control personnel

movement within a cleanroom facility. For example, including
separate entries and exits for cleanroom personnel. This ensures
that cross contamination between staff, garments, etc. is kept to a
minimum and also allows control of the disposal of cleanroom
garments which have been used in the cleanroom. The same principle
should also be applied to materials used in the process. Production
materials should arrive at the critical zone via one path and the
finished products leave via a different path. There should also be a
separate path for waste materials in order to avoid contamination
between raw materials, finished products and waste. This may in
practice involve complex transfer hatch or airlock designs. However,
the benefits of keeping pathways separate will certainly outweigh
the costs.

Once designed, there are certain parameters that need to be met.

Overarching the design concept is the air system, which is referred
to as HVAC systems. HVAC of the required standard is achieved
 Designing, Commissioning and Risk Assessing Cleanrooms 231

through air-handling units. Air handling units are subdivided into

a supply air handling unit and an extract air handling unit. The
supply air handling unit is responsible for establishing the required
temperature and humidity levels as well as preliminary air filtration
and, together with the extract air unit, for air circulation. Air handling
units are assembled from a sequence of components:

• Dampers for adjusting or controlling air flow rates to the

required levels.

• Ventilators for maintaining the air in circulation.

• Air coolers, employing chilled water as coolant, for reducing the

air temperature as well as for condensing excessive water out
from humid outside air.

• Droplet separators for removing eventual droplets entrained

into the supply air from the wet surfaces of the air coolers.

• Humidifiers for increasing the relative humidity of excessively

dry outside air if required.

• Sound absorbers for reducing the noise generated by the

ventilators to an acceptable level.

• Supply air filters positioned plus extract air filters if required.

Extract air grilles positioned close to the floor or – if particle
dissemination in the room is low – close to the ceiling or
integrated into it capture the extract air which is then carried
via extract air ducts to the extract air handling unit from where
it is partially exhausted into the outside atmosphere and partly
recirculated into the supply air unit.

• Air heaters operated with hot water for increasing, if necessary,

the air temperature; in cold climates, additional pre-heaters may
be necessary if the first air filter stage requires protection against
supercooled water droplets or snow borne by the outside air.
232 Cleanroom Contamination Prevention and Control

Also, of importance is deciding on the number of air inlets, for the

filtered air to enter the cleanroom and the number and position of
diffusers. In cleanrooms, the supply of clean air to critical areas can
be improved by the choice and positioning of diffusers. The main
variable that influences airflow and local particle concentrations in
cleanrooms is the method of air supply. According to Whyte and
colleagues (2012), when an inlet has no diffuser, the air supply gives
a pronounced downward airflow jet to the floor, which reduces the
penetration of particles into the air stream. The cleanest conditions
in the cleanroom are found below the inlet, but poorer conditions
tend to be observed in most other parts of the cleanroom. A four-
way diffuser has been shown to give much better mixing of the air
throughout the cleanroom and hence a more even concentration of
particle contamination. A related consideration is with the positioning
and number of room extracts, which influences air exchange rates.

In addition to the overall design of the airflow pattern it is

important to consider local airflows around equipment, instruments
and personnel. Although the room design may be more than
adequate for its class when at rest, the addition of staff and production
equipment and may change the airflow pattern significantly. It is
also necessary to avoid stagnant areas where the air is refreshed too
slowly, or eddies that recirculate contaminated air without taking
it away. The number, spacing and type of supply air diffuser and
room extracts are therefore important aspects of design.

Each of these components is critical to the design concept.

Other ways by which contamination is controlled within clean-

rooms are:

• Staff enter and leave through airlocks and wear protective

clothing such as hats, face masks, gloves, boots and coveralls.

• Equipment inside the cleanroom is designed to generate minimal

air contamination. There are even specialized mops and buckets.
Cleanroom furniture is also designed to produce a low number
of particles and to be easy to clean.
 Designing, Commissioning and Risk Assessing Cleanrooms 233

• Contamination control requires personnel to practice aseptic

techniques; to wear specially designed clothing; and to behave
in ways which will minimize contamination.

• Common materials such as paper, pencils, and fabrics made

from natural fibers are excluded from the cleanest working
areas (including areas used for aseptic filling).

• Cleanroom HVAC systems also control the humidity to low

levels, such that extra precautions are necessary to prevent
electrostatic discharges.

• Opportunities for contamination are minimized (such as not

leaving water puddles on the floor).

• Cleanrooms should be regularly cleaned and disinfected using

approved techniques and validated disinfectants (verifying
contact time and concentration, through surface studies and a
supporting field trial).

These other mechanisms are noteworthy, but they fall outside the
scope of this chapter.

COMMISSIONING A NEW CLEANROOM

As indicated above, for guidance on the commissioning of clean­
rooms the ISO 14644 series of standards provide a useful starting
point. Again it is important to emphasize that not all of the parts
are of relevance to pharmaceuticals and healthcare (instead these
apply to areas like micro-electronics)The most applicable parts are
(Sandle, 2016):

• ISO 14644 Part 1 (2015), which covers the classification of

air cleanliness in cleanrooms and associated controlled
environments. Classification in accordance with this standard is
specified and accomplished exclusively in terms of concentration
of airborne particulates.
234 Cleanroom Contamination Prevention and Control

• Part 2 (2015) specifies requirements for periodic testing of a

cleanroom or clean zone to prove its continued compliance
with ISO 14644-1 for the designated classification of airborne
particulate cleanliness.

• Part 3 (2005) specifies test methods for designated classification

of airborne particulate cleanliness and for characterizing the
performance of cleanrooms and clean zones. Here there is a
difference between unidirectional airflow and turbulent airflow.

• Part 3 (2001) addresses airflow velocity, airflow volume, air

pressure difference, installed filter leakage, airflow visualization,
recovery (clean-up), containment leakage (isolators).

• Part 4 (2004) is aimed at those who purchase, specify and design

cleanrooms. The standard specifies requirements for the design
and construction of cleanroom and clean air devices, as well as
requirements for start-up and qualification.

• Part 5 (2004) provides the basic requirements for operating and

maintaining cleanrooms and associated controlled environ-
ments. It includes sections on training, installation of equipment
and the types of equipment used in cleanrooms.

• Part 6 (2004) is a series of definitions.

• Part 7 (2004) looks at isolators, gloveboxes, RABS and other

separative devices.

• Part 14 (2016) is about defining the quality of materials going

into cleanrooms and it is of pharma interest.

Note: At the time of writing, Part 4 is under revision. ISO standards

are version controlled by the year of issue, and the reader should
verify the in-use version by checking either the ISO website or the
website of the appropriate national standards organization.
 Designing, Commissioning and Risk Assessing Cleanrooms 235

14644 Part 1 describes cleanroom classification. Here classifi-

cation (read: commissioning, qualification or certification) is the
process of qualifying the cleanroom environment by the number
of particles using a standard method. The outcome is to determine
the classification of cleanroom according to standards, e.g., Room x
is ISO class y. Once a cleanroom has been fully classified there are
certain aspects that need to be maintained such that the cleanroom
operator can continue to claim that the cleanroom continues to meet
the classification and in which state that classification was made.
This is where the second part of the ISO 14644 standard comes into
play. Cleanroom classification needs to be repeated on a frequency
defined by ISO14644-2 (Sandle, 2017a).

An important element of the cleanroom qualification or

commissioning exercise is with planning. There are several stages
required, which are discussed below.

Validation Master Plan

A Validation Master Plan (VMP) is a document that details the
main commitments and decisions that need to be made. The VMP
is a document that documents the way the company will operate,
who has control over the various aspects of the validation activities,
and how production, quality control, and personnel management
will be directed. The VMP will contain the core elements of a User
Requirements Specification (URS), and the specifications from the
URS will need to be captured into the VMP. Depending on the scale
of the project, Validation Plans (VPs) may be produced from the VMP.

From these plans the Design Qualification (DQ), Installation

Qualification (IQ), the Operational Qualification (OQ), and the
Performance Qualification (PQ) need to be produced and authorized.
All of these functions must be detailed in the VMP. Within the
qualification documents, responsibilities need to be detailed and job
functions nominated.
236 Cleanroom Contamination Prevention and Control

Factory Acceptance Testing

Factory Acceptance Testing (FAT) is undertaken by executing a suite
of documented tests on a completed system or item of equipment.
Each individual test, known as a case, exercises a particular operating
condition of the user’s environment or features of the system,
and will result in a pass or fail. The test environment is usually
designed to be identical, or as close as possible, to the anticipated
user’s environment, including extremes (such as temperature and
humidity). These test cases must each be accompanied by test case
input data or a formal description of the operational activities (or
both) to be performed – intended to thoroughly exercise the specific
case – and a formal description of the expected results. In terms of
the cleanroom, the FAT is more applicable to the clean air device.

Design Qualification
The Design Qualification is a document which compares the URS
and the Proposed Design. This is usually executed in conjunction
with input from the end users and the design company. The
document additionally confirms what will be produced or tendered
will meet the URS and design intentions and provides formal
procedures/protocols for determining compliance.

COMMISSIONING (QUALIFYING) CLEANROOMS

The commissioning process starts when the installation of the
cleanroom is completed. That is a process usually performed
between the contractor and the client as a witness, where all parties
evaluate the cleanroom parameters using approved tables. The
intention is to show how much of the design is fulfilled concerning
the requirements, performance, operation and maintenance.

Some of tests which are performing during commissioning

period are (Sandle, 2017b):
 Designing, Commissioning and Risk Assessing Cleanrooms 237

• Airflows and air-changes.

• Differential pressures.
• Duct leakage tests.
• Interlocks.
• Temperature and relative humidity.
• Unit performance.
• Assessment of microbial levels.

It is essential that all cleanrooms to be qualified by personnel

familiar with all requirements and procedures for certain tests
to be performed. It is additionally important for the personnel
involved in the qualification tests to present unbiased reports
with a summary of the test results and where appropriate, with
conclusions and recommendations.

Different qualification tests within one cleanroom are performed

during its lifetime. These consist of:

Installation Qualification
The Installation Qualification ensures that the installation of the
equipment is accurate, where possible, by comparing the existing
system against the design and the manufacturer’s specifications and
it is providing a complete documentary record of that installation.

The IQ ensures that the components marked on the Piping and

Instrumentation Diagrams (P&IDs) are installed as described; that
HVAC instrumentation measuring devices are calibrated; and that
the installation of the wall, ceiling and lighting systems (that is the
cleanroom in general) is according to the specification.

The IQ Protocol must contain the following information:

238 Cleanroom Contamination Prevention and Control

• Information on equipment services.

• Identification of mechanical and electrical components.
• Equipment information and documentation.
• Manufacturers/suppliers calibration records.
• Drawing information.
• Verification of correct location.

Operational Qualification
The objective of the Operational Qualification is to check those
operational aspects of the HVAC system that are deemed critical
to its satisfactory performance and compare the data obtained with
approved specifications and Good Manufacturing Practice (GMP).
Prior to this, it should be confirmed that all critical instruments have
been calibrated for performing the OQ tests. When assisting the end
user during the validation of predetermined specifications with the
OQ a contractor must determine whether the system corresponds
to the manufacturer’s description and installation requirements. In
doing so, the reproducibility of the operating characteristics can be
evaluated, thus verifying that adequate control of the air handling
system can be routinely exercised. The tests detailed within the OQ
Protocol will have been specifically requested by the client.

Table 1 The procedures for Qualification tests

Test Procedure described within the

ISO 14644-3:2005 can be found in:
Airflows and Air-change rate Test Section B.4
Differential Pressure Test Section B.5
Temperature and Relative Humidity Test Section B.8 and Section B.9
Filter Leakage Test Section B.6
Airflow Visualisation Test Section B.7
Recovery Test Section B.12
Particle Counting Test Section B.1
 Designing, Commissioning and Risk Assessing Cleanrooms 239

ISO 14644-3:2005 provides the procedures for the OQ tests and

details how they are to be performed:

There are also other tests described within the standard ISO
14644-3:2019 such Electrostatic and Ion Generator Test, Particle
Deposition Test and Containment Leak Test which fall outside the
scope of this book.

Performance Qualification
The Performance Qualification (PQ) is performed once the OQ has
been completed within the time specified between the client and the
contract tester. For the PQ not all of the tests undertaken in the OQ
need to be performed. Here a risk-based approach can be taken, such
as only considering those tests deemed critical and which can show
that the system can run continuously. With the PQ tests it is essential
to demonstrate that the HVAC system is capable of maintaining
parameters such airflows and air-changes, differential pressure,
temperature and humidity within the acceptance criteria, i.e.,
demonstrates that the process or equipment performs as intended in
a consistent manner over time.

MICROBIAL CONTROL
Missing from the ISO standards is the development of the microbial
environmental monitoring program and the importance of
undertaking a more intensive form of initial monitoring in order to
establish a baseline. Both US FDA and EU GMP (particularly the
latest draft of Annex 1 in place at the time of writing) recommend
extensive microbial monitoring to establish the performance of a
cleanroom or suite of cleanrooms (FDA, 2019; EU, 2020).

By gathering as much data as possible over a representative

period of time, such as 2–3 months then the level of control can
be established, appropriate monitoring limits can be set and other
measures, such as appropriate cleaning and disinfection can be
established. In terms of the time period, the US FDA recommends
240 Cleanroom Contamination Prevention and Control

between three and six weeks, although a longer time period may be
required depending upon the consistency of the trend data.

In terms of the purpose of the monitoring, it is:

• To monitor cleanrooms at a sufficient frequency in order

collect enough data to be able to examine trends across time,
occupancy, shifts and operations. As indicated above the data
should be collected across several weeks, ideally taken each day,
and should ideally produce at least 100 data items. These data
can be used to assess the state of microbiological control of an
environment.

• The monitoring should reflect the cleanroom environment

under representative conditions when cleanrooms are occupied
and equipment is running).

• Data collection will relate to the numbers of microorganisms

and the incidence of detection using pre-defined monitoring
limits). At the end of the assessment, these data should be used
to set operational alert and action levels.

• In addition, some of the microorganisms recovered should be

characterized and trended. This analysis will help with the
assessment of the cleaning and disinfection regime.

• The data can also be used to show whether contamination levels

increase through manufacturing as the process, in relation to
adjacent cleanrooms. The contamination cascade should be
designed so that the environment becomes cleaner as the process
moves downstream.

• Depending on the purpose of the cleanroom, the data may

help to assess the risk to the environment and potentially to
the product. This is enhanced by selecting monitoring locations
which are meaningful when product risk is considered.
 Designing, Commissioning and Risk Assessing Cleanrooms 241

• When action levels are exceeded or adverse trends are detected

appropriate investigations must be performed using docu­
mented procedures to determine the contamination source, the
impact upon the product and to set corrective or preventative
actions. For this risk based methodologies can be deployed.

• The data review will also help the facility to understand the
performance of the people and equipment, and the suitability of
operating protocols.

• Ultimately, the data provide information about environmental

control and to the effectiveness of the initial cleanroom design in
relation to the type of operation.

In most circumstances, the collected data and its associated analysis

will need to be provided to a regulatory authority as part of the
change process.

There are different sources of microbiological contamination

within clean environments: water, air, surfaces (both within the
room and from equipment) and personnel. These hazards should
be evaluated by the microbiologist in terms of the relative risks
to the product, and the environmental monitoring programme
should be orientated towards the points of greatest risk. The
sampling methods should be appropriate in relation to the types
of contamination sources (and a comprehensive monitoring
programme should assess each of the main contamination
sources). The greatest risks are those which could lead to product
contamination. This is illustrated in the diagram below (Figure 1).

The primary sources of contamination are people and water.

This is because both are vectors of contamination. People are the
most significant source of contamination, although they are a highly
variable and unpredictable source. Microorganisms are shed from
hair, skin, eyes and mucous membranes. Microorganisms are either
deposited into the air stream or can spread through contact. Water is
a common feature in pharmaceutical processing (as an ingredient, a
242 Cleanroom Contamination Prevention and Control
Figure 1 Diagram showing sources of microbial
contamination within a cleanroom

cleaning agent, a diluent for disinfectants, steam supply, and so on).

The concern with water in cleanrooms is that it not only provides a
means for microorganisms to survive, it provides the opportunity for
the numbers of microorganisms to increase and microorganisms are
invariably found in all residues of water (some bacteria, especially
Gram-negative rods, can grow and multiply in low nutrient states).

The secondary sources of contamination are air and surfaces.

The air in most areas contains microorganisms. However, the
number of microorganisms will vary according to the cleanroom
grade. Air is a secondary contamination source because air is a
vector for microorganisms, but it is not a nutritive environment and
whilst some bacteria can survive in air streams they cannot multiply.
Generally Gram-positive bacteria are more commonly found in
air (typically Bacillus spp, Staphylococcus spp, and Micrococcus
spp) (Ackers and Agallaco, 2001). Bacteria in air are normally
in association with dust particles or skin flakes, rather than as
individual microorganisms (for which the term “microbial carrying
particle” is sometimes used). This makes the microorganisms
heavier and more prone to gravitational settling. Therefore, what
often matters most is not the microorganisms in the air but their
potential for settling. A well-designed cleanroom will filter air (to
dilute the number of microorganisms) and have a pressure cascade
 Designing, Commissioning and Risk Assessing Cleanrooms 243

to prevent re-contamination of a clean area from a less clean area

(since microorganisms cannot move against an air current) (Whyte
and Eaton, 2004).

The other secondary contamination source is materials and

surfaces. Here, the key risks are the transfer of items in and out of
a clean area, where materials are more at risk if they are of a design
that cannot be easily cleaned or disinfected; and from personnel
touching surfaces. Another risk is the contamination of surfaces
through deposition (such as settling from the air) (Sandle et al., 2010).

Based on these contamination sources certain factors will lead to

contamination risks being more likely. These factors include:

• Poorly designed cleanrooms.

• Water remaining on surfaces for prolonged periods.

• Inadequate cleaning and sanitization.

• Inadequate personnel gowning.

• Poor aseptic practices such as direct surface-to-surface

transfer (such as by personnel directly touching the product or
contaminated water entering the process).

• Airborne transfer, often arising from personnel shedding

microorganisms. Shedding increases with increased personnel
movement and fast movement also increases the potential for
microbial dispersion.

Based on the above, the locations selected and the methods deployed
should reflect a consideration of these hazards. This should be
established through a documented risk assessment.

This should feed into the environmental monitoring program

in order to assess the cleanroom under performance qualification.
This program should be documented. As a minimum, the program
should address the following elements:
244 Cleanroom Contamination Prevention and Control

• Types of monitoring methods, such as:

– Active air-sampling: volumetric air-sampler.
– Passive air-sampling: settle plates.
– Surface samples: contact plates and swabs.
– Personnel samples: finger plates and gown plates (for
aseptic processing areas).
– Particle counting is performed using a discrete particle
counter.

• Culture media and incubation conditions.

• Frequency of environmental monitoring:

– This will probably be daily, and spanning across a minimum
of three weeks and up to 12 weeks, in order to capture a
sufficient range of activities and operations, and to generate
a sufficient number of data points (ideally >100).

• Selection of sample sites (where monitoring will take place):

– To determine these, a study of the room (layout and
equipment) and the process (understanding what happens,
what equipment is used and what the people working in
the area do) is required. It is important that the types and
locations for monitoring have relevance to the process; that
the data produced must is meaningful (Lowry, 2001). This
is often achieved by mapping the process and the flow of
people and materials, by applying risk assessment tool such
as HACCP (Jahnke and Kuhn, 2003).

• Maps showing sample locations.

• Duration of monitoring.

• When and where the samples are taken (i.e., during or at the
conclusion of operations).
 Designing, Commissioning and Risk Assessing Cleanrooms 245

• Method statements describing how samples are taken and

methods describing how samples are handled.

• Clear responsibilities describing who can take the samples.

• Processing and incubation of samples.

• Alert and action levels.

• Method of data analysis, including trending.

• Investigative responses to action levels excursions.

• Appropriate corrective and preventative actions for action level

excursions.

The output of the PQ phase is a report that analyzes the performance

of the cleanroom using specified parameters for the physical
aspects and using a detailed data review for the microbial aspects.
Importantly, the PQ is a pre-requisite for the final certification of the
newly built or modified cleanroom.

In developing the biocontamination assessment for the PQ, it

is important to spend time on design and with getting everything
right. For example, if the locations selected are unrepresentative
or the frequency is insufficient, or if the microbial identification
method is inaccurate, during the performance qualification, there is
a false sense of control is achieved and this will lead to the need to
remediate contamination at a later stage and this brings with it a risk
of regulatory citation.

Re-qualification or re-commissioning
Re-qualification tests refer to the period of time during which it is
obligatory to conduct the tests defined in the ISO 14644-2 (2015)
for cleanrooms in order to prove there is continued compliance
between the cleanroom performance and the requirements set in
246 Cleanroom Contamination Prevention and Control

the standards. For re-qualification a number of tests are required

(Sandle and Saghee, 2013):

• The Particle Counting Test is required to confirm if the

classification of the cleanroom remains within the required ISO
classification. The frequency of re-classification is:
– ≤ISO 5 on every six months.
– >ISO 5 on every 12 months.
A risk-based case can be made to carry out ISO class 5 areas every
12 months, should data support such a longer time interval.

• The Airflow Volume or Airflow Velocity and Differential

Pressure Tests are tests which must be performed during the
period of 12 months.

• Other optional tests recommended to be conducted within a

period of time of 24 months are:
– Filter leakage test (except when a new filter is installed than
the test should be performed immediately).
– Airflow visualization (required for some unidirectional
airflow devices within cleanrooms).
– Recovery test.
– Containment leakage test.

The frequency intervals for performing tests are the following:

• If the test must be performed on every six months the average

interval must not exceed 183 days and the test not to be
performed for more than 190 days.

• If the test must be performed on every 12 months the average

interval must not exceed 366 days and the test not to be
performed for more than 400 days.
 Designing, Commissioning and Risk Assessing Cleanrooms 247

• If the test must be performed on every 24 months the average

interval must not exceed 731 days and the test not to be
performed for more than 800 days.

TESTS REQUIRED FOR THE COMMISSIONING

AND QUALIFICATION OF CLEANROOMS
This section of the chapter examines the tests required for commis-
sioning, qualifying and re-qualifying cleanrooms.

HVAC system component installation check

This test is part of commissioning and/or IQ process. Here “as built”
drawings have to be challenged with the installation. Components
have to be checked for any visible damage and any visible impurities.
Using the latest revision of the ductwork distribution drawing(s)
and HVAC schematics to be completed tables:

Table 2 Example of table which register basic drawings data

required for this test

Drawing No: Description Revision Date Confirm provided

Initials Date
XXXXX XXXXXX XX

COMPONENTS VERIFICATION

It needs to be verified that the “Components” in the selected section

of the distribution ductwork and installed clean room elements
are correct (suitably located, installed securely and free from
damage) When this is completed the drawing has to be identified
by writing title. Drawings at the end should be signed or initialled
with the date.
248 Cleanroom Contamination Prevention and Control

Examples of components are shown in Table 3.

Table 3 Examples of cleanroom component testing

Constant Volume Control Boxes Sockets, switches, lights

Ductwork Terminal diffusers, grilles
Volume control dampers and motorized Panels, doors
dampers
Sensors (pressure sensors, pressure switches, Air handling units, coils, humidifiers,
temperature and humidity sensors etc.) dehumidifiers, inverters
Diffusers and grilles Pumps, valves, pipes, instruments
Filters (EU4, EU9, H14, U15) Sockets, switches, lights etc.

Airlock test
Airlocks may be used to control the entry of personnel or materials
to a secure area such as a cleanroom; the airlock is composed of two
doors that are electrically interlocked in such a way that the two
cannot be opened simultaneously. For performing the airlock test it
is important to identify and check the interlock systems within the
facility. The test starts with using the latest revision of the Air Lock
Signalization layout, where it is confirmed that interlocking systems
have been installed in the required location, supplied with power (or
compressed air where applicable, usually in fire rated areas), cables
(or pipes where compressed air) are properly connected and tested,
and the semaphores are installed together with the emergency button.

The objective is to determine if the airlock doors are operating

according specifications hence the acceptance criteria will be to
identify if the airlock system operates as per manufacturer and
design specifications.
 Designing, Commissioning and Risk Assessing Cleanrooms 249

Common interlock tests are designed to:

• Verify that room interlocked doors cannot be opened simultan-

eously.

• On approach to door, button should be pressed or swipe card

(where applicable) to gain access.

• LED lights will change from red to green.

• Entry into room/airlock sensors (where applicable) will detect

presence of personnel and once the person has passed through
doors, closer will activate to close doors (applicable only where
doors are semi-automatic or automatic).

Fan commissioning test

This test is typical for the period of commissioning after system is
balanced. The purpose of this test is to give identity to the installed
fan section of the air handling unit (motor, fan, pulleys, belt, power,
etc). This test also has target to confirm the spare capacity of the
motor/fan.

Air filtration
HEPA filters are used to provide clean air to the cleanroom. HEPA
filters are replaceable, extended-media, dry-type filters in rigid
frames with set particle collection efficiencies. The filters are designed
to control the number of particles entering a clean area by filtration.
In ISO 14644 class 5/EU GMP Grade A zones HEPA filters also
function to straighten the airflow as part of the unidirectional flow.
In order to measure the effectiveness of the filters they are checked
for leaks. Leakage is assessed by challenging the filters with a particle
generating substance and measuring the efficiency of the filter.
250 Cleanroom Contamination Prevention and Control

In terms of design, a high efficiency filter is made of a sheet of

highly specialized glass paper folded back and forth over spacing
devices, and then sealed into a frame, which is usually extruded
aluminum. This construction allows a very large filter area to be
contained within a relatively small frame. Several types of high
efficiency filter are available. Mini-pleat filters are typically between
24 mm and 75 mm deep and are therefore very compact. Deep
pleat filters are usually between 150 mm and 300 mm deep and can
therefore contain much more filter paper than a mini-pleat with the
same face area. This means that for the same face area and volume
flow rate the pressure drop is much lower. Cartridge high efficiency
filters, which are cylindrical, are suitable for some applications. The
performance of a high efficiency filter is defined by its percentage
efficiency or percentage penetration at the most penetrating particle
size (MPPS) for that filter.

Most cleanrooms and isolators use H14 class HEPA filters, which
is designed to remove 99.997% of particles from the air of a size
0.3 µm and greater. At their rated volume, these have a maximum
overall penetration of 0.005% at the MPPS and a maximum local
penetration (i.e., for a single leak) of 0.025%. The MPPS is usually
between 0.1 µm and 0.2 µm. It is very important to note that, if the
volume flow rate through the filter is greater than the rated volume,
then the performance of the filter deteriorates rapidly.

HEPA filters function through a combination of different aspects

(Xu et al., 2010):

• First, there are one or more outer filters that work like sieves
to stop the larger particles of dirt, dust, and hair. Inside those
filters, there is a concertina – a mat of very dense fibers – which
traps smaller particles (sometimes called sieve effect).

• Second, the inner part of the HEPA filter uses different

mechanisms to catch particles as they pass through in the
moving airstream. At high air speeds, some particles are caught
and trapped as they smash directly into the fibers, while others
 Designing, Commissioning and Risk Assessing Cleanrooms 251

are caught by the fibers as the air moves past (sometimes called
the interception effect). At lower air speeds, particles tend to
wander about more randomly through the filter (via Brownian
motion) and may stick to the fibers as they do so (sometimes
called the inertia effect). The inertia effect is effective above all
for particles > 1 μm. Due to their comparably high mass inertia
these particles will be unable to follow the air streamlines as
they are deflected around the fiber, and thus will tend to collide
with the fiber.

• A further mechanism is the diffusion effect, which is of relevance

for very small particles (below 0.5 μm) with a correspondingly
low mass. This is as a result of the constant collisions with the
molecules of the gas surrounding them, these particles experience
an irregular diffusional movement around their streamline (with
the oscillations becoming more pronounced with decreasing
particle diameter), thus increasing the probability that the
particle will collide with one of the fibers of the filter.

Both the interception and the inertia effect become more effective
with increasing particle diameter. The opposite is true for the dif­
fusion effect: mobility of the particles and thus diffusion and the
pro­bability of capture will increase with diminishing particle size.
Once deposited on the surface of the fiber, the particles will remain
fixed there by surface forces (van der Waals forces).

Together, these mechanisms allow HEPA filters to catch particles

that are both larger and smaller than a certain target size. The sum
of these mechanisms is referred to as integral separation efficiency.

HEPA filters are protected from blockage by pre-filters which

remove up to about 90% of particles from air. Air filter systems for
cleanrooms normally are composed of three stages:

• First filter stage: A medium performance fine dust filter for

protecting the air handling unit against soiling.
252 Cleanroom Contamination Prevention and Control

• Second filter stage: A high performance fine dust filter for

maintaining the air ducts in a clean state.

• Third filter stage: HEPA filter for guaranteeing the quality of the
supply air entering the working rooms.

To use an example, if air contains about 3 × 108 particles per m3, and
there is one pre-filter and one HEPA filter:

• Pre-filter leaves about 3 × 107 per m3 as a challenge to the HEPA

filter.
• The terminal HEPA filter leaves about 103 per m3.
• In EU GMP this is within the limit for Grade “at rest” (and thus
to ISO 14644 class 5).

An alternative measure is the integral particle penetration. This

is the percentage of particles passing through the filter. In order
to measure the effectiveness of the filters they are checked for
leaks. Leakage is assessed by challenging the filters with a particle
generating substance and measuring the efficiency of the filter.

There are higher grade alternatives to HEPA filters. These are

ULPA (Ultra Low Penetration Air) filters. These grades of filters are
not common to pharmaceuticals and healthcare and are more often
found in the electronics industry.

Installed filter leakages

The risk of HEPA filter leakage is of importance and must be
measured in an appropriate way. Filter leakage refers to the filters
that supply air directly into the cleanroom (normally HEPA filters
within the pharmaceutical sector). This is an important assessment
for, should a HEPA filter leak, then an excess level of particles could
enter the room. The purpose of performing regular leak tests is to
ensure the filter media, filter frame and filter seal are free from leaks.
 Designing, Commissioning and Risk Assessing Cleanrooms 253

Leakage is checked by challenging each filter with an aerosol

of particles dispersed upstream of the filter and scanning over
the downstream face to ensure that there are no leaks that exceed
a specified level of penetration. The aerosol selected for HEPA
leak testing should not support microbial growth and should be
composed of a sufficient number or mass of particles. Guidance is
provided in EN 1822-1:2019.

The challenge aerosol presented to the upstream side of the filter

should be stable, homogeneous and have a concentration of between
20 µg/L and 50 µg/L in accordance with ISO 14644-3:2019. For a
stable test the aerosol providing particles should have the following
distributions:

• More than 20% by mass of particles less than 0.5 µm.

• More than 50% by mass of particles less than 0.7 µm.
• More than 75% by mass of particles less than 1.0 µm.

When performing this task, it is important to ensure that the aerosol

supply is homogenous. To maintain homogeneity, the aerosol
should be injected into an upstream duct at a distance that is at least
15 duct diameters from the upstream face of the filter.

Leaks are assessed though the use of scanning equipment, the

most common of which is an aerosol photometer. The photometer is
used to measure the upstream aerosol concentration as well as the
downstream penetration of the HEPA filter by the aerosol. By the
use of light scattering instrumentation, upstream and downstream
particle concentrations can be measured. In essence, if 10,000 0.3
micron sized particles are blown into a HEPA air filter, only 3
particles are allowed to pass through. This would give the filter a
99.97% efficiency at 0.3 micron rating

Filters that can be face scanned are relatively straightforward to

test. However, filters that are inaccessible or housed within complex
installations, are difficult to measure and require specialist contractors.
254 Cleanroom Contamination Prevention and Control

Ductwork leakage testing

Duct leakage tests are assessments of air leakage to confirm that
what has been designed, and installed on site, has minimum losses
as possible of air which prevent mainly in saving energy, achieving
airflows, temperature and so on. These are not direct leak tests of the
HEPA filters.

There are three classes of ductwork concerning pressurization.

• Class A: up to 500 Pa positive and maximum negative 500 Pa.

• Class B: up to 1000 Pa positive and maximum negative 750 Pa.
• Class C: up to 2000 Pa positive and maximum negative 750 Pa.

In relation with the ductwork classification system, a risk based

approach can be adopted as follows:

• If the ductwork is classified as high pressure ducts – all ductwork

is tested.

• Where medium pressure class 10% of random selected ducts

will be tested.

• Low pressure ducts are untested, only tested if there is agreement

between the customer and the installers.

Leak testing starts with defining the area needs to be tested, and
marking at the drawings. Fan has to be provided with enough
capacity to achieve the required pressure which is defined for testing.
With flexible connections then connect to the ductwork. All open
ends on the ductwork need to be blanked. Manometer Gauge has to
be provided at the test rig to read initial pressure. Flow instrument
needs to be provided for reading the leakage. Test to be maintained
for approximately 15 minutes. All details from the test should be
recorded and signed.
 Designing, Commissioning and Risk Assessing Cleanrooms 255

Other ways how to find leaks include:

• By looking: any visual damages on the ducts.

• By listening: when leak appears noise is significant at that place.

• By feeling: running your hand over the area (helps if the hand
is wet), and this is more recommended for supply ducts (when
positive pressure is tested).

• Soap and water: is a visual test, where target is to find bubbles at

the duct surface.

• By generating smoke inside ductwork.

Airflows and air-changes

Some of the most important tests conducted in the cleanrooms are the
examination of airflows and air change rates. The purpose of these
tests is to measure the supply/extract airflow rate in the cleanrooms
and clean zones and to confirm the design specification for the air
changes per hour. Effective airflows are essential in order to reach
the desired cleanliness levels, as well as the required temperature
and relative humidity levels within cleanrooms. Within cleanrooms
the air is normally operating at a turbulent flow (this is where air
enters the room with non-uniform velocity). With clean air devices,
the object is to have unidirectional airflow.

ISO 14644 defines turbulent flow as:

”Airflow not running in one direction. Air distribution, during which

primary air entering the cleanroom or clean area is mixed with the air
inside the room by means of induction.” (ISO 14644-1:2015)

The filtered clean air is introduced into the cleanroom with a

swirling effect and generates an increasing dilution of the particle
concentration. With this flow principle, the staff’s behaviour
according to cleanroom conditions is particularly important in
order to ensure the required airborne particulate cleanliness class.
256 Cleanroom Contamination Prevention and Control

Unidirectional airflow (occasionally referred to as low-turbulence

displacement flow or imprecisely as “laminar airflow”) is defined
by the same standard as:

“Unidirectional airflow, regulated airflow with uniform speed across

the entire cross-section of a cleanroom or clean area, which is regarded
as parallel airflow.” (ISO 14644-1:2015)

Each cleanroom grade has a set number of air changes per hour.
Air changes are provided in order to dilute any particles present
to an acceptable concentration. Any contamination produced in
the cleanroom is theoretically removed within the required time
appropriate to the room grade. This is important because particles
would otherwise build up in enclosed spaces if there is no ventilation.

The air-changes per hour are expressed through a mathematical

formula which examines the airflow volume (m3/h airflow) supplied
to a given room (m3). With this:

• The airflow enters in the Air Handling Unit (AHU) with a certain
velocity (m/s) which directs this airflow through the fan and the
ductwork to the terminal diffusers in the cleanroom.

• The airflow velocity (m/s) × the surface of the ductwork or the

terminal filter (m2) = airflows (m3/s).

• To calculate the airflow in m3/h one should simply multiply the

value of m3/s × 3600.

• The volume of the room is given in m3 as a result of multiplying

the room surface (m2) by the room height (m).

• The air changes per hour are expressed as:

Airflows (m3/h)
Air changes per hour (1/h) =
Volume (m3)
 Designing, Commissioning and Risk Assessing Cleanrooms 257

Tables are available which show the values of air changes per hour
(from-to) that are needed in order to attain a certain cleanliness
level in a given industry. It’s worth mentioning that the designer
should pay attention to the following issues, regardless their partial
accuracy:

• The purpose of the given room.

• The filtration level.
• The number of operators.
• The heat gains from the equipment, etc.

The most appropriate method for measuring airflow rate at the inlet
is:

• First select the correct size hood to fully cover the filter or
terminal outlet/inlet.

• Select the volume program on the processor program.

• After covering the terminal outlet hold the hood on the

downstream face of the supply diffuser and measure air volume
in m³/h.

• Read the indicated volume directly from the instrument and

recorded. That volume presents actual air per terminal ref
diffuser.

To calculate room total air which is summary of actual air supply

for all terminal diffusers within the cleanroom follows the
following formula:

Σ Room TOTAL Air =Terminal Ref1 +Terminal Ref2 …..+Terminal Refn.

Then calculate:

Σ Room TOTAL Air (m³/h)

Air Changes per Hour (1/h) =
Room Volume (m³)
258 Cleanroom Contamination Prevention and Control

It should be noted that the flow hood recordings are not always
accurate and that is why many manufacturers provide a table of the
k (correction) factor, which is directly influenced by the airflow rate.
The calculation of the k – factor should be taken in consideration
whilst performing measurements. This is affected by the air passing
through, the dimension of the diffuser, the shape of the diffuser,
whether a terminal filter is installed and so forth.

As a worked example, consider a cleanroom with area of

87.4 m2 and height of 2.8 m has designed to achieve 14ACH. At
the ceiling are six terminal filters which bringing conditioned
and filtered air from one central air handling unit located at the
building plantroom. Each terminal according the design to achieve
the required classification and microclimate conditions is estimated
that supplying 600 m3/h of air.

Room Volume: Room Area × Room Height = 87.4 × 2.8 = 244.72 m3

Table 4 Table with calculation of the percentage of the actual air

entering within the cleanroom

Terminal ref. Design air (m3/h) Actual air (m3/h) Percentage of design (%)
1 600 605 100.83
2 600 627 104.50
3 600 573 95.50
4 600 597 99.50
5 600 612 102.00
6 600 622 103.67
TOTAL 3600 3636 101.00
Note: Percentage of design (%) calculation: Actual Air/Design Air × 100

Σ Room TOTAL Actual Air (m³/h) = 3636

Actual Air Changes/hour =
Room Volume (m³) 244.72

Actual Air Changes / hour = 14.85(1/h)

 Designing, Commissioning and Risk Assessing Cleanrooms 259

Supply airflow rate calculated from filter face velocity

To meet ISO 14644 measurements of the airflow, velocity should be
at approximately 150mm to 300mm from the filter face (it should
be noted that for the FDA Guide to Aseptic Processing the location
should be risk assessed by the use, and to meet EU GMP the
measurement should be taken at the “working height”). In order
to comply with these different standards additional measurements
may need to be taken).

According ISO 14644-3:2019 the number of measuring points

should be sufficient to determine the supply airflow rate in
cleanrooms and clean zones. This should be the square root of 10
times of area in square meters. However, not less than four readings
should be taken. At least one point per filter should be measured.
More positions for measuring are better because in that way is
determining the uniformity of the air in front of the filter.

Equipment used for airflow measurements includes the use

of a flow hood. This is an instrument capable of measuring total
supply or extract air volume, the instrument may have various
combinations of frame sizes and hoods and can therefore cover
a large number of grilles or filter housing sizes. The instrument
works on the Wilson grid principal of air measurement, and
involves the collecting hoods gathering the full volume of air and
passing it over the Wilson grid. The pressure readings taken across
the grid are fed into the meter and this produces a direct readout
of the volume either in metric units.

Air-patterns and air-movement

Whilst HEPA filters provide air of a low number of particles, this is
not sufficient in itself to ensure that the cleanroom remains at low
level of contamination because contamination can be generated by
the people and activities in the room. Therefore, air dilution and air
movement are important.
260 Cleanroom Contamination Prevention and Control

Most rooms are turbulent flow areas. This is where it is con­

sidered sufficient to dilute the concentration of contaminants
disseminated in the room by supplying it with sufficient quantities
of HEPA filtered air according to the principle of turbulent mixing
airflow. For higher and highest air cleanliness requirements a
different airflow pattern must be employed: unidirectional airflow.
Unidirectional airflow areas are used for higher cleanliness states
(such as aseptic filling) and they use far greater quantities of air than
turbulent flow areas.

Airflows for critical activities (such as aseptic filling), need to

be studied in order to show that air turbulence does not interfere
with critical processes by mapping smoke patterns. Airflow patterns
are reviewed by generating smoke and capturing the air pattern
as a motion picture. The object is to examine the aerodynamics to
determine if there are vortices and zones with stagnant air; such
areas may indicate where contamination could occur. Such results
should feed into the environmental monitoring program.

Airflow velocity and direction

ISO 14644 class 5/EU GMP Grade A zones (unidirectional airflow
devices in ISO 14644 class 7/EU GMP Grade B rooms) have a
requirement for a controlled air velocity and unidirectional air
flow (either horizontal or vertical). These are monitored using an
anemometer.

Unidirectional airflow is normally established by means of a

continuous HEPA filter ceiling which will cause the airflow to move
on more or less parallel streamlines and with a reasonably uniform
velocity and very little lateral mixing. Thus, this airflow pattern
achieves removal of particles and microorganisms disseminated
into the clean zone air by the most direct path. Thus, the air velocity
is designed to be sufficient to remove any relatively large particles
before they settle onto surfaces (Ensor and Foarde, 2007). The
requirement for air velocity for the manufacture of sterile products,
as required by both FDA and within Europe, is ±0.45 meters per
 Designing, Commissioning and Risk Assessing Cleanrooms 261

second (Eudralex, 2014; FDA, 2004). There are, however, differences

in the regulations in relation to where airflows are measured.

The FDA guidance on sterile products requires airflow

measurements to be taken at 6” from the filter face and “proximal to
the work surface” (FDA, 2004). The EU GMP Guide requires readings
to be taken at the working height (Eudralex, 2014). Working height is
defined in local procedures and demonstrated as effective by way of
airflow (smoke) studies. The practice at many organizations, during
bi-annual re-qualifications, is that airflows will be measured from
both locations using the specification detailed in the table above.

In addition to qualifications, many organizations elect to meas-

ure airflows periodically before commencing an activity.

Air changes
Each cleanroom grade should have a requirement for a set number of
air changes per hour (the air exchange rate). To put this into context,
a typical air-conditioned office will have something between two
and 10 air changes per hour in order to give a level of comfort. The
number of required air changes in a cleanroom is typically much
higher (as a rule, a minimum of 20 and often far higher). Air changes
are provided in order to dilute any particles present to an acceptable
concentration (thus air change is a way of expressing the level of air
dilution which is occurring).

Differential pressures
In order to maintain air quality in a cleanroom the pressure of a given
room must be greater relative to a room of a lower grade. This is to
ensure that air does not pass from “dirtier” adjacent areas into the
higher-grade cleanroom. Thus, the differential pressure is one of the
main characteristics to be considered in every cleanroom. Usually
cleanrooms are exposed to a higher pressure than the pressure of
their surroundings. Very often a differential pressure is required
262 Cleanroom Contamination Prevention and Control

between different cleanrooms (a cascade of differential pressures).

This will depend upon the different cleanliness levels required.

This test is conducted in order to prove that the air system is

capable to maintain differential pressures between cleanrooms, and
cleanrooms and their surroundings in a given period of time. During
the measurements of the differential pressure all doors which belong
to inspected area must be closed.

Cleanroom differential pressures are normally set at between

5 and 25 Pa as this allows doors to be opened and overcomes
problems for cleanroom operators in relation to the high pressure
difference, which arises due to air leakage. Air leakage arises due
to “gaps”. Such gaps are mainly due to the uneven floors and
the impossibility of a door gasket entirely covering in an uneven
surface. Furthermore, if certain parts of the cleanroom have not been
siliconized such irregularities can increase the “air flow” noise and
reduce the comfort for the operators working inside the cleanroom.

The differential pressure check is conducted after any air

balancing of the cleanrooms has been completed. Some rooms/areas
are more or less pressurized than the others and by comparing the
differences differential pressure is determined. In order to attain
the required room pressure, it is necessary to determine the volume
of air flow entering into the cleanroom and the volume of air
leaving the cleanroom. For instance, in a perfectly sealed room the
straightforward explanation is when, for example, a cleanroom has
1000 m3/h air entering the room, measured with an instrument fixed
on the distributive element and 1000 m3/h air leaving the room, also
measured at the distribute element at the exit. With this example,
the cleanroom is neither positively nor negatively pressurized. In
contrast, where the supply air volume in the cleanroom is higher than
the air volume which is extracted from the cleanroom, then room
is positive pressurized. Conversely, where a room has a negative
pressure this means the supply air volume in the cleanroom is lower
than the extracted air volume. In reality such ideal conditions are not
always achieved because the supply and the extract of air (treated or
 Designing, Commissioning and Risk Assessing Cleanrooms 263

untreated) from the room does not always passes through diffusers
and grilles and often leaks through the gaps which are existing
within one cleanroom. In short, all cleanrooms leak to a certain
extent. What matters is how big the leak is and whether the required
pressure differential be maintained.

Cleanrooms should be equipped with differential pressure

gauges which enable continuous monitoring of the differential
pressures and provide an opportunity to the persons in charge of
monitoring of the performances of the cleanroom to react in case of a
drop in pressure. The instruments which are used for determining the
differential pressures are the electronic micromanometer, inclined
manometer, or alternative other differential pressure gauges can be
used.

Most cleanrooms are fitted with magnahelic gauges which serve

for measurement of the differential pressures between two rooms.
A magnahelic gauge has two ports: one port is located within the
cleanroom with higher pressure (plus (+)) and one located in the
cleanroom which is designed to have lower pressure (minus (–)).
The difference between these two cleanrooms gives the differential
pressure. Depending on the accuracy of the magnahelic gauge, which
is based on its scale, the pressure differences can vary considerably.
When the range is wider the instrument is less sensitive.

The digital manometers for differential pressures are significantly

better than magnahelic gauges as they can be more easily fitted, be
located in areas which allow easier maintenance and because they
can provide continuous monitoring. Such devices can be set in
the cleanrooms, on the technical floor above each room or at any
other suitable place. Normally these manometers are connected to
a BMS (Building Management System) where the cleanroom user
has a clear display of the overall state of the system and the of the
differential pressure influence between the rooms.

The following differential pressure between the cleanrooms is

considered as acceptance criteria:
264 Cleanroom Contamination Prevention and Control

• The acceptance criteria will be ≥5 Pa between areas of the same

ISO Class according ISO 14644-1.

• The acceptance criteria will be ≥15 Pa between areas with

different ISO Class according ISO 14644-1.

Sometimes within cleanrooms differential pressures fall out of the

specification. In such events, checks should be undertaken to assess:

• Check if the doors are properly closed.

• Check the gasket under the door. If the gasket is properly closed
(this is means assessing whether the room was properly sealed
as before the discrepancy happened).
• Check if something is blocking any supply or extract point
within the room.
• Check the neighboring rooms if there is anything unusual with
the pressures.
• Check and compare with the previous measurements the supply
air flow for the cleanroom.
• Check and compare with the previous measurements the supply
air flow for the air handling unit.
• Check the dampers on the extract are there on the same position
like during the commissioning time.
• Check the frequency of the unit.
• Check the set point of the velocity sensors, or the differential
pressure sensor located in the supply duct.
• Check the pressure drop through the filters on the air handling
unit and terminal filters if any.
 Designing, Commissioning and Risk Assessing Cleanrooms 265

Particle classification
The particle classification test is the ultimate determinant of whether
a cleanroom meets its expected classification (e.g., ISO class 5, 7 or 8).
This consists of undertaking the following steps.

Particle sizes to be measured

For pharmaceuticals and healthcare, to meet FDA regulations,
particles of a size ≥0.5 μm are required to be measured. For those
wishing to classify using ≥5.0 μm size in addition, the standard
requires that the outcome is expressed using a macro-particle size
descriptor, namely:

ISO M (20;≥ 5.0μm); LSAPC

Where:
M = macroparticles.
20 = class limit (value taken from EU GMP Annex 1).
≥ 5.0 μm = the particle size under consideration.
LSAPC = Light scattering airborne particle counter (reference
to the test instrumentation).

For EU GMP a consideration is with the inclusion of the ≥5.0 μm

size, which is required for routine assessment. In such cases:

Grade A
ISO 5; at rest, operational; ≥0.5 μm.
ISO M (20; ≥5.0 μm); at rest, operational; LSAPC.

Grade B
ISO 5; at rest; ≥0.5 μm, and M (29; ≥5.0); at rest, LSAPC.
ISO 7; operational; ≥0.5 μm, 5.0.
266 Cleanroom Contamination Prevention and Control

Number of locations to be measured within the cleanroom

The method for selecting the number (and position) of particle
counter locations within a cleanroom is based on a look-up table.
The table uses a range of cleanroom sizes and provides the number
of locations required (if the exact room size is not listed, the user
selects the next largest room size and picks the appropriate number
of locations). These numbers are based on a statistical method called
hypergeometric distribution, based on particles not being normally
distributed. The approach allows each location to be treated
independently.

Location of particle counters within the cleanroom

Once the number of locations has been selected, the room is divided
up into sectors and a particle counter placed in each sector. The
position where the counter is placed within each sector is determined
by the user. The standard allows counters always to be placed at the
same point within the sector; randomly placed within the sector;
or evenly distributed; or selected by risk. The risk-based approach
would be the best one to adopt. A risk-based decision could be based
on variables like: room layout; equipment type; airflow patterns;
position of air supply and return vents; air-change rates; and room
activities.

Generally, simply selecting the room center should be avoided.

The reason for not selecting the center of the location relates back to
the issue of particle distribution: particle counts no longer assumed
to be homogenous within a sector. Furthermore, additional locations
can be added at the discretion of the facility. This might arise from
the room-by-room risk assessment.

Volume of air to be sampled

The volume of air sampled needs to be sufficient to detect at least 20
particles of the largest particle size selected. The standard requires a
minimum of 2 liters per location; the application of the formula can
result in this being higher. Generally, the lower the particle count
 Designing, Commissioning and Risk Assessing Cleanrooms 267

limit, the greater the volume to be sampled (so a larger volume is

taken from an EU GMP Grade B room compared with a Grade C
room). When operating a particle counter in unidirectional airflow,
e.g., Grade A, the counter probe must be orientated into the airflow
or pointed upwards for turbulent flow air.

Assessing results
For each location within the cleanroom, each individual result must
comply with the intended class limit. Assessment of results involves:

• Recording the results for each location.

• Converting the results to a particles per cubic metre.

• This is set out using the formula published in the standard.

• It should be noted that the formula states “number of particles

at each location or average”. This is because an option exists
to add more than one particle count location per sector. When
this occurs the results are averaged and the average used as the
number to proceed with the above calculation. Individual results
may fall outside of the class, provided that the mean is within.
• Although assessment is based on an average, each individual
result must be within limits. Those out of limits need to be
investigated.

Should out of limits results be obtained an investigation should be

performed into the origin of the particle counts. This should conclude
consideration of room design, equipment, and operator activities.
Once a root cause has been identified, and corrective measures put
in place, the exercise should be repeated. A risk assessment will be
required to assess activities performed in the cleanroom between
classification exercises.

Test certification
In terms of requirements for test certificates in relation to cleanroom
classification. Certificates should state:
268 Cleanroom Contamination Prevention and Control

• Name and address of the testing organization.

• Date of testing.
• Number and year of the publication of the relevant part of ISO
14644, e.g., ISO 14644: 1–2015.
• Location of cleanroom (or clean zone).
• Specific representation of locations, e.g., diagram.
• Designation of cleanroom.
• ISO class (plus EU GMP).
• Occupancy.
• Particle count sizes considered.
• Test method used (and any departures or deviations).
• Identification of test instrument and calibration certificate.
• Test results.

Clean up times (or “recovery test”)

Connected to air changes is the time taken for a clean area to return
to the “at rest” condition, appropriate to its grade, in terms of
particulates. This is referred to as the clean-up time and it is assessed
by calculating the recovery rate. For this test, airborne particles are
generated to a level that exceeds the class for the room. The time
taken for the room to reduce the particles to a level back within
the required class is then measured. The standard target is for a
cleanroom to achieve this within 15–20 minutes.

Airflow visualization
Airflow visualization is sometimes referred to as an air-pattern or
smoke study, and it is applicable to some unidirectional airflow
devices (such as those used for aseptic processing). The test involves
blowing smoke around the cleanroom to ensure that the flow is
 Designing, Commissioning and Risk Assessing Cleanrooms 269

satisfactory (with aseptic filling this is the air moving away from the
critical zone). The air pattern, visualized with the smoke, is assessed
for undesired movement such as counter drafts, stagnant areas,
areas where air rises (especially after striking a surface), turbulent
areas (within areas that are designed to have unidirectional airflow)
and cross currents (Sandle et al., 2017).

Separation concept and positive pressure differentials

Connected to the measurement of air flow is positive pressure. In
order to maintain air quality in a cleanroom the pressure of a given
room must be greater relative to a room of a lower grade. This is
to ensure that air does not pass from “dirtier” adjacent areas into
the higher-grade cleanroom (this can also be observed by smoke
studies). Generally, this pressure differential 15–20 Pascals. The
most commonly encountered problems relate to situations when
cleanroom doors are opened and here it can be difficult to maintain
pressures.

Pressure differentials form a key part of the separation effect.

This is where a cleaner area is separated from a less clean area. The
main of separation effects are:

• Displacement concept: the spill-over of clean air into areas of

lower air cleanliness level, providing an aerodynamic barrier.
This is mainly used when an area, protected by unidirectional
airflow, is to be separated from a surrounding area of lesser air
cleanliness level with turbulent airflow.

• Pressure concept: the maintenance of a pressure differential

between zones of different air cleanliness levels.

• Physical barrier concept: where an impervious physical barrier,

such as where a wall prevents contamination transfer to a clean
zone from a less clean area.
270 Cleanroom Contamination Prevention and Control

Other factors
For certain cleanrooms, temperature, humidity and lighting require
control, either because of a process step or as a means to minimize
contamination. Lighting should be adequate, uniform and anti-glare,
to allow operators to perform process tasks effectively. A range of
400 to 750 lux is recommended.

With temperature and humidity, the objective is for cleanroom

operators to feel comfortable. Furthermore, to comply with EU
GMP, for Grade B areas, where the temperature must fall within
18±3°C. This helps to minimize the risk of operator particle
shedding, in relation to the wearing of the cleanroom suit, as well as
for maintaining a comfort factor.

ENVIRONMENTAL MONITORING
As indicated above, environmental monitoring should be risk based
and the output from the performance qualification may necessitate
an update. This may include a review of the locations for monitoring
determined using HACCP or equivalent process flow approach.
With this locations are orientated to the points of greatest personnel
activity or where there is exposed product. In terms of the frequency,
this often continues at a relatively high level and then (ideally)
decreases as the data improves. Both the cleaning and disinfection
regime and the environmental monitoring regime can help shape
each other; with the pattern of viable monitoring results helping
to set the frequency of cleaning and disinfection and the rotation
between different biocides (Sandle, 2012b).

Environmental monitoring should be risk based, with the

locations for monitoring determined using an appropriate tool, such
as HACCP or equivalent process flow approach (De Vecchi, 2014).
With this, locations are orientated to the points of greatest personnel
activity or where there is exposed product. In terms of the frequency,
this often starts at a relatively high level and then (ideally) decreases
 Designing, Commissioning and Risk Assessing Cleanrooms 271

as the data improve (Deschenes, 2008). Both the cleaning and dis­
infection regime and the environmental monitoring regime can
help shape each other; with the pattern of viable monitoring results
helping to set the frequency of cleaning and disinfection and the
rotation between different biocides (Sandle, 2012b).

RISK BASED APPROACH TO

CLEANROOM ASSESSMENTS
Risk is important within the context of this chapter for two reasons.
Risk needs to be considered in terms of running and maintaining
cleanrooms and risk can be used to help to streamline some of
the elements of the design, commissioning and re-commissioning
process. Some risk considerations have been mentioned in the
above sections; the objective of this section is to discuss some areas
where risk can be used to optimize cleanrooms so that efficiencies
can be sought and contamination control maintained.

With risk, in the context of this book it is important to keep in mind

the microbial risk factors, where changes to the scope or frequency
of testing could lead to environmental or product contamination.
There are different ways to conceptualize, such as a combination of
the probability of occurrence of harm and the severity of harm. More
specifically in the pharmaceutical context, risk could be: “An event
in the production, control and supply of a drug which has potential
an adverse health effect” (WHO, 2011). Risks can be either intrinsic
or extrinsic. Intrinsic risks are those which are an integral part of
manufacturing system or building design having influence on quality
of product. This includes the design of cleanrooms or some error
with their design or commissioning that leads to a contamination
event occurring. Extrinsic risks are those that come from outside the
manufacturing process such as from the external environment or
personnel. In the cleanroom context, this could be air ingress from a
less clean area or with the less predictable aspects of human behavior.
272 Cleanroom Contamination Prevention and Control

Design factors
With design, there is a view held by some cleanroom experts that
many aspects of cleanroom operations are over-specified, leading
to energy waste and adversely contributing to carbon emissions.
This includes HEPA filters, which are often of a higher specification
than is necessary. This is because the majority of viable particles
are in fact carried on comparatively large non-viable particles,
such as skin flakes, and smaller viable particles, such as spores,
are dealt with, very comfortably at an efficiency which is at worst
the “most penetrating particle size” efficiency of the grade of filter
selected (Whyte et al., 2013). Relating to this area of potential over-
specification is air supply and air exchange rates. It is often that
many cleanrooms have excessive air supply that is associated with
high capital and running costs, and energy waste. While there is
scope to alter this care must be taken, for a low air supply may
result in too high a concentration of contamination, and major
remedial work to rectify the problem (Whyte et al., 2016).

Hence an important aspect of design to review is with air-

exchange rates. First, there is a regulatory difference arises with air
change rates. Here the FDA Guidance (2004) requires 20 air changes
per hour whereas EU GMP (2014) no longer specifies a rate, using
the term appropriate air change rates instead. Depending on the
inspectorate, a justification may be required for the set air-change
rates. However, in practice air change rates above 20 are generally
accepted by most regulators without the need for additional
exposition. Higher air change rates equate to higher airflows and
more energy use. For lower grade cleanrooms, many users have opted
for air change rates of around 15 air changes per hour. However, for
critical areas air change rates are normally considerably in excess
of 20 and far higher again for areas of higher particle generation
like changing rooms. Another option is to vary air exchange rates
at different times, depending on room occupancy states. In most
cleanrooms, people are the primary source of contamination
(Reinmüller, 2001). Once a cleanroom is vacated, lower air changes
per hour to maintain cleanliness are possible, allowing for setback
of the air-handling systems. Variable speed drives can be used on
 Designing, Commissioning and Risk Assessing Cleanrooms 273

all recirculation air systems allowing for air flow adjustments to

optimize airflow or account for filter loading. On this basis, for
energy conservation reasons, airflow of the ventilation systems may
be reduced to low levels during non-operating periods. Energy
savings in cleanrooms can be realized by reducing air changes.
To assess the risk, strict monitoring of air quality and cleanliness
of cleanroom components is important to assure the environment
is still suitable for the operations carried out. However, if systems
are turned off the potential for unacceptable room contamination to
occur must be risk assessed.

A further design risk factor is with air velocities, which are

applicable to unidirectional airflow devices. The optimum velocity
for unidirectional airflow has been accepted as 0.45 m/s and indeed
the EU GMP guidelines specify a guidance value of 0.36 to 0.54 meters
per second. However, some tests have shown that lower airflow
velocities are almost as effective and can give substantial energy
savings. A reduction in airflow velocity needs to be supported by
airflow visualization (“smoke studies”) and through particle count
assessments.

Hence, design and commissioning can be applied not only to

ensure appropriate biocontamination control, but also to maximize
performance of targeted energy efficiency measures, as well as to
save energy in cleanrooms where no particular effort has previously
been made to utilize energy efficiency strategies. Commissioning in
particular can be used as an effective strategy for managing energy
use, costs, and associated greenhouse gas emissions for cleanroom
construction and operation.

Risks of cleanrooms failing can be minimized by positioning the

outside air intakes at sufficient height above ground and flat roofs,
at sufficient distance from exhaust air outlets and known biocon-
tamination sources in the vicinity of the site, with due consideration
of the predominant wind directions. A further protective measure is
by ensuring adequate outside air filtration.
274 Cleanroom Contamination Prevention and Control

The biocontamination risk inherent in the humid elements of

air handling units can be controlled by ensuring efficient drainage
of the water condensed out of the air stream; by positioning
droplet separators downstream of air coolers; and by air filtration
downstream of all dissemination sources of microorganisms (Prabu
et al., 2017).

Commissioning and re-commissioning

With the commissioning and re-commissioning process itself, risk
can help to review the tests and to help to decide whether all of
the tests are required and with setting the frequency for retesting.
Assessment is also required after certain events. For instance, a
change of terminal HEPA filters or a significant stand-still of the
HVAC system. For assessing cleanrooms and related air systems for
risks, arguably the most appropriate risk assessment tool is a Failure
Mode and Effects Analysis (FMEA) procedure. FMEA is more suited
to engineering systems (Whyte and Eaton, 2004).

When such an exercise is run, the scope of commissioning

works can be reduced, supported by a justification. Typically, as a
minimum a classification exercise should examine:

• The airborne particle count.

• The installed filter system leakage test for verification that

terminal HEPA filters and their support structure are free from
leaks.

• Airflow visualization for demonstrating by means of flow

marking with smoke that particles are successfully swept away
from critical zones as well as for analyzing, for example, the
airflow in the vicinity of equipment.

• The recovery test for demonstrating how quickly a room in

which a high level of particulate contamination had been
generated returns to its original state of air cleanliness.
 Designing, Commissioning and Risk Assessing Cleanrooms 275

Of these, particle counting is of great importance for assessing

microbiological control. As a minimum, classification is confirmed in
the in-operational state by taking non-viable particulate readings at
a defined number of locations for ≥0.5 µm size particles (sometimes,
within Europe, ≥5.0 µm are additionally assessed. This is due to
particles of this size and larger being mentioned within EU GMP,
although EU GMP itself permits only ≥0.5 µm to be assessed provided
that ≥5.0 µm are assessed for routine monitoring in relation to sterile
products manufacture) (Eudralex, 2014).

Further with particle counting, the ISO 14644 standard discusses

the three occupancy states. “As built” only refers to newly constructed
cleanrooms; however, the “static” and “occupied” states ostensibly
relate to any established cleanroom when it comes around for re-
commissioning. Is classifying in both states actually required? On a
risk basis, the most important factor is for cleanrooms to be classified
once in use, in the occupied or “in operation” state as this presents
the bigger risk – the room with people in it and equipment running.
From a risk based perspective, the static state would only require
monitoring should the occupied state fail the particle assessment,
with static monitoring used for troubleshooting to help to determine
the cause of the particle counts (for example, a further failure in the
static state could suggest a problem with a HEPA filter, whereas a
pass in the static state might suggest a problem with the room clearing
up sufficiently with a given level of personnel occupancy or from an
item of equipment contained within the room generating particles).

An area which requires a risk-based perspective is in relation to

energy saving, where changes can be sought from the original design
concept for older facilities or incorporated for new builds. Reducing
the power to cleanrooms is something that many companies are
seeking. In relation cleanrooms the desire to save on energy is often
for economic reasons or to minimize the contribution to global
warming of the atmosphere. Whilst energy reduction measures
are laudable, process safety and microbiological risks must always
predominate. Thus, energy saving and optimization in clean facilities
must never interfere with process requirements. The best means to
276 Cleanroom Contamination Prevention and Control

achieve energy savings is by adopting a stepwise approach, such as

making small adjustments, gathering key data (especially particle
levels) and assessing this before seeking a further adjustment.

Savings to the operation and hence classification of cleanrooms

can be made with the use of isolators. Such devices also confer
advantages in terms of contamination control when undertaking
critical activities like aseptic filling. A lower classification can be
used because with isolator, as discussed above, the isolator is a
minienvironment that uses a physical barrier (usually a plastic film,
plastic sheet or glass) to isolate the susceptible or critical part of
the manufacturing process from the rest of the room. The critical
manufacturing area is kept within the isolator and provided with
large quantities of the very clean air, the rest of the room being
provided with lower quantities of air.

Maintaining cleanrooms
In terms of maintaining cleanrooms (or on-going compliance),
although cleanrooms provide the structure for a contamination
control program, it should also be noted the cleanrooms, where
im­properly maintained, can contribute to contamination risks. An
assessment of cleanrooms should form part of any risk assessment
that contributes to the contamination control strategy. When
cleanrooms go wrong, they can contribute to the distribution of con­
taminants borne by the outside air throughout a building. Further­
more, cleanrooms can propagate airborne cross-contamination
by particles of pharmaceutically active substances, processed in
parallel within the facility, from room to room. In addition, where
humid elements fail (air coolers and humidifiers), this raises
the potential for the multiplication of microorganisms and their
subsequent entrainment into the supply air. Verification of control
is provided through an appropriately designed and well executed
environmental monitoring program, assessing the levels of particle
and microbial contamination.
 Designing, Commissioning and Risk Assessing Cleanrooms 277

With each of the above considerations, the enthusiasm to alter

the operation of HVAC parameters could impact upon the level
of non-viable particles and viable counts. It is therefore important
that microbiologists are consulted in any decisions relating to
cleanroom parameters.

SUMMARY
The importance of well-maintained and functioning cleanrooms
cannot be underestimated. Cleanrooms are an essential feature
of contamination control and are required to reduce the risk to
pharmaceutical products by minimizing the transference of micro­
organisms. How well cleanrooms function is a product of their
design and how well they have been commissioned. The process of
commissioning requires an assessment of physical parameters (most
of which are adequately described in the ISO 14644 standards, noting
regulatory differences in terms of grades and particle concentrations)
and microbial levels (which are not adequately covered by ISO
standards and for which regulatory expectations should be noted).

The main focus of the chapter has been on airborne con­

tamination and how design and operation of cleanrooms helps to
control this risk. In reducing airborne risks, priority should be given
above all to actions which aim at impeding particle generation and
dissemination. This includes:

• Process improvement towards reduced particle generation.

• Improving machines and process equipment towards reduced

particle dissemination, such as through construction measures
and via suitable selection of construction materials.

• Maintaining enclosures around dust-generating process steps.

• Capturing of dust at source of origin by means of suction or

combined blowing/suction devices.
278 Cleanroom Contamination Prevention and Control

The contamination control task can become focused on the control

of the airborne contaminants which remain after the above steps
have been exhausted. For controlling these remaining airborne
contaminants, several important elements are required to interact:

• Air filtration of the supply air and, where necessary, also extract
air prior to exhausting it into the external atmosphere.

• Appropriate airflow patterns for controlling particles dissemin-

ated in rooms.

• Effective procedures for separating areas with different air

cleanliness requirements from each other thus impeding
cross-contamination.

• Prevention of uncontrolled infiltration of outside air through the

building shell into air cleanliness controlled areas.

• Good aseptic behaviors.

• Cleaning and disinfection regimes.

With the focus on air, it should not be underestimated the probability

of microbial carrying particle settling onto surfaces (especially those
particles of a large diameter or where there are inadequate air
patterns). This means that effective cleaning and disinfection regimes
are also required to help to maintain biocontamination control.
Increased knowledge of these parameters assists with developing a
risk based approach for the testing that is required, especially when
it comes to re-qualification. As technologies advance in terms of
real-time data gathering, greater inferences can be drawn from data
to help to determine the extent, scope and frequency of the testing
required to support re-certification.

In outlining the requirements for cleanroom construction and

design, and for the continued monitoring, the chapter has shown
cleanrooms and clean air devices are an essential topic for those
concerned with contamination control.
 Designing, Commissioning and Risk Assessing Cleanrooms 279

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particulates and microbials.” In Esteves, S.C., Varghese, A.C.,
and Worrilow, K.C. (Eds.) Clean Room Technology in ART Clinics:
A Practical Guide. CRC Press, Boca Raton, USA. pp. 75–91.

Sandle, T. (2016) “ISO 14644 Parts 1 and 2 – The revised cleanroom

standard and contamination control.” In Madsen, R.E. and
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Environmental Monitoring: a Comprehensive Handbook. Volume 7.
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 Designing, Commissioning and Risk Assessing Cleanrooms 283

Sandle, T. (2013a) “Contamination Control Risk Assessment.” In

Masden, R.E. and Moldenhauer, J. (Eds.) Contamination Control
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PDA, River Grove, IL, USA. pp. 423–474.

Sandle, T. (2013b) Sterility Testing of Pharmaceutical Products. DHI /

PDA: Bethesda, MD, USA. pp. 129–160.

Sandle, T. (2013c) “Contamination Control: Cleanrooms and

Clean Air Devices.” Encyclopedia of Pharmaceutical Science and
Technology. Fourth Edition, Taylor and Francis: London. pp.
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Sandle, T. (2012b) “Application of Disinfectants and Detergents in

the Pharmaceutical Sector.” In Sandle, T. The CDC Handbook: A
Guide to Cleaning and Disinfecting Cleanrooms. Grosvenor House
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Sandle, T., Budini, M., Rajesh, T. (2017) “Airflow studies and airflow
mapping.” In: Sandle, T. and Saghee, M.R. (Eds.) Cleanroom
Management in Pharmaceuticals and Healthcare. Euromed
Communications: Passfield, UK. pp. 361–376.

Sandle, T., Saghee, M.R. (2013) “Cleanroom certification and

ongoing compliance.” In: Sandle, T. and Saghee, M.R. Cleanroom
Management in Pharmaceuticals and Healthcare. Euromed
Communications: Passfield, UK. pp. 169–184.

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Monitoring and Cleanrooms. IDMA-APA Guideline, Technical
Monograph No. 5. Indian Drug Manufacturers Association,
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industry: An overview. Journal of Drug Delivery and Therapeutics
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of cleanrooms by people. European Journal of Parenteral and
Pharmaceutical Sciences 15(3): 73–81.

Whyte, W. (1986) Sterility assurance and models for assessing

airborne bacterial contamination. Journal of Parenteral Science and
Technology 40: 188.

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airborne cleanliness in non-unidirectional airflow cleanrooms.
European Journal of Parenteral and Pharmaceutical Sciences 21(2):
38–43.

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deposition in cleanrooms: Deposition mechanisms. Clean Air
and Containment Review Issue 24, pp. 4–9.

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high efficiency air filters against microbe-carrying particles
(MCPs) in cleanrooms. Clean Air and Containment Review Issue
14, pp. 4–9.

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cleanroom with respect to air supply inlets. Clean Air and
Containment Review Issue 10, pp. 4–9.

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from people. European Journal of Parenteral and Pharmaceutical
Sciences 12(2): 39–46.

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for use in risk assessment during pharmaceutical production.
 Designing, Commissioning and Risk Assessing Cleanrooms 285

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11–15.

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Practices: main principles for pharmaceutical products. Annex 3,
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sterile room) – Restricted Access Barrier System (RABS). Pharm.
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ABOUT THE AUTHOR

Tim Sandle is Head of Microbiology, Risk Management and Sterility
Assurance at BPL. He is also a visiting lecturer at the University
of Manchester and University College London. A member of the
Pharmig committee since 2002, Tim has run a Pharmaceutical
Microbiology website since 2010. Tim has written or edited 30 books,
165 peer reviewed papers and 450 technical articles pertaining to
microbiology, healthcare and pharmaceutical processing.
 9

CLEANROOM COMMISSIONING
AND QUALIFICATION

Wei Sun
Engsysco, Inc.
Farmington Hills, MI
USA

Commissioning and qualification for cleanrooms are necessary

procedures related to manufacturing of pharmaceutical and
biotechnology products in clean process environments. Each
procedure is to make sure that a facility with all equipment will
function as required and be approved by the regulatory agencies
that have jurisdiction over that facility (FDA, 2004). The goal of
the commissioning and qualification process is to obtain a quality
product manufactured in this facility. For finished products, this
quality means the product has been consistently produced with the
proper identity, strength, and purity to ensure safety and efficacy.
Quality also means the manufacturer has quality systems in place
and has performed a risk management assessment of the facility and
the manufacturing processes for the product. While commissioning
can be considered as primarily an engineering effort with necessary
checks and tests, qualification is often under supervision from the
quality assurance team (ASHRAE, 2019; Sun et al., 2017).

287
288 Cleanroom Contamination Prevention and Control

COMMISSIONING SUPERVISION
Commissioning should be a precursor to system qualification
and process validation (WHO, 2007; ASTM, 2013). Cleanroom
commissioning is a systematic and documented approach from
startup until turnover of facilities, systems, and equipment to end-
users (FDA, 2004; WHO, 2007). The outcome of commissioning
efforts ensures that user requirements and design specifications are
met. Commissioning verifies that what was specified was installed,
that it functions properly, and that it was successfully turned over to
the users. Cleanroom commissioning activities may include design
reviews, Factory Acceptance Testing (FAT), site acceptance testing
(SAT), and functional testing (ISPE, 2001). During commissioning,
the operations and maintenance manuals are verified, and personnel
are trained. The summary reports are generated with any deviations
encountered during testing. FDA guidelines require that user
requirements should be traceable throughout the lifecycle, each
commissioning team is required to test traceability of all design
specifications for all facilities, process equipment and software
developed for the project.

A Good Manufacturing Practice (GMP) pharmaceutical

cleanroom performance and effectiveness is required to be tested,
certified, commissioned and qualified to meet validation require­
ments. However, cleanroom performance testing (CPT) (NEBB, 2009)
could be conducted at different stages/phases at various facilities.
In some pharmaceutical process facilities, the cleanrooms are tested
for “as-built” condition and tested again for “operational” condition
after the balancing of heating, ventilation, and air conditioning
(HVAC) systems, during the later commissioning phase, there could
be no additional CPT tests but only to check if previous test results
meet user requirements and design specifications. Facilities are
required to have independent CPT tests during the commissioning
or qualification phase to ensure that CPT test results are under
updated and optimized process conditions.

Detail cleanroom CPT testing standards can be found in the most

recent ISO and NEBB standards. ISO 14644-3 (2019), Cleanrooms
 Cleanroom Commissioning and Qualification 289

and Associated Controlled Environments – Part 3, Test Methods

outlines the procedures for testing the performance of clean spaces.
Besides, NEBB’s Procedural Standards for Certified Testing of
Cleanrooms (2009) also provides very detailed procedures for indoor
environmental testing which can be used as a basis for testing,
certification, commissioning, and qualification of cleanrooms.

Typical testing for commissioning of cleanrooms could be the

same tests that are used for cleanroom performance testing and
certification, but the commissioning efforts should also include extra
verifications and documentations under final process conditions,
which may include (IEST, 2015; 2016; ISO, 2019; NEBB, 2009):

• Airborne particle concentration

For room classification (classified pharma spaces), typical
particle range is from 0.1 µm to 5.0 µm, while 0.3 µm and 0.5 µm
are the most common sizes to be monitored and controlled.
The equivalent air cleanliness in ISO Class can be calculated.
Ultrafine particles (smaller than 0.1 micron) and macroparticles
(larger than 5 micron) are not very common in pharma test.

• Airflow intensity
In unidirectional applications, measure supply airflow velocity
and uniformity, and calculate the supply airflow volumetric
rate and air change rate. In non-unidirectional applications,
measure airflow volumetric rate directly and calculate air
change rate.

• Airflow direction and visualization of airflow patterns

Airflow direction can be demonstrated to determine the basic
cleanroom airflow patterns in many critical areas/spots inside
a cleanroom in its at-rest and operational states. The airflow
patterns can be conducted by Computational Fluid Dynamics
(CFD) simulations and animations during design stage, and
by smoke test with video documentation after the cleanroom
space is constructed and becomes operational and functional to
validate the effectiveness of the design concepts.
290 Cleanroom Contamination Prevention and Control

• Temperature and humidity

Verify the air temperature and relative humidity levels are
within the control limits over the time period specified by the
customer for the area being tested.

• Room pressure differential

Verify the capability of the cleanroom air control system to
maintain specified pressure differential between the cleanroom
and its surroundings. For a classified space (as in sterile product
manufacture), verify if the specified pressure differential value
(at least 10 Pa typically) can be established between two adjacent
rooms of different air cleanliness classes.

• High-efficiency particulate air (HEPA) filter system integrity

Confirm that final HEPA filter system is properly installed
without leakage and defect through scan test.

• Containment leak
Determine if there is intrusion of unfiltered air into the clean-
room from outside the cleanroom enclosure through joints,
seams, doorways and ceiling panels. It is particularly important
where potent materials are exposed to the room environment.

• Recovery time
Determine whether the cleanroom is capable of returning to
a specified cleanliness level within a finite time, after being
exposed over an agreed period of time to a source of airborne
particulate challenge.

• Electrostatic and conductivity

Evaluate electrostatic voltage levels on objects, static-dissipative
properties of materials (such as floors, workbench tops, etc.) and
the performance of ion generators in eliminating static charges
on surfaces. Both tests are used for electrostatic control.
 Cleanroom Commissioning and Qualification 291

• Particle deposition
Verify the quantity and size of particles deposited onto settling
plates for colony forming units (CFU) counting over an agreed
period of time as a measure of surface contamination.

• Segregation
Assess the separation effectiveness achieved by a specific
airflow pattern, challenging the less-clean area with high
particle concentration and determining the particle migration
in the cleanroom side of the segregation. The segregation
mechanism could be a cleanroom door in either closed or
opened conditions, access door on separator, room enclosure,
or demarcation bench across clean and dirty sides in a gowning
room (Sun et al., 2017; ISO, 2019).

Commissioning should also include the setting up, balancing,

adjustment and testing of the environmental control systems, to
ensure that it meets all the requirements, as specified in the user
requirement specification and capacities as specified by the designer.
Acceptable tolerances for all system parameters should be specified.
The installation records of the system should provide documented
evidence of all measured capacities of the system, schematic
drawings, protocols and reports. The data should be contained
in the operating and maintenance (O&M) manuals for any future
changes and upgrades to the system. Training should be provided
to personnel after installation of the system (Sun et al., 2017; WHO,
2007; ASTM, 2019).

QUALIFICATIONS
After the commissioning is completed, the qualification process
can start. Commissioning is a prerequisite of the qualification
process. Qualification is primarily concerned with verifying and
documenting that the facility, systems and process equipment that
have a direct impact on product quality are rigorously tested and
documented. Qualification is a systematic, quality-based approach
to ensuring and documenting, thereby demonstrating that facilities,
292 Cleanroom Contamination Prevention and Control

building systems, and process equipment are suitable, perform

as specified in project design documents, and will deliver every­
thing required for safe and repeatable drug products, including the
facility design, installation, operation, maintenance, documentation,
and pharmaceutical processing, filling, capping, holding, handling,
and storage (ASHRAE, 2019 ; Sun et al., 2017).

Validation and qualification are essentially components of the

same concept. The term qualification is normally used for equip-
ment, utilities and systems, and validation for processes. In this
sense, qualification is part of validation (WHO, 2007).

The primary goal of qualifications is to deliver safe and

effective products to patients. Therefore, it is necessary that the
environmental control of the facility making that product function
is at its specified, and ideally optimal performance (ASTM, 2013).
Maintaining environmental control of complex facilities and
processes is significant and will require the integration of industrial
automation products, for the process equipment, and building
control systems such as HVAC as well as environmental monitoring
systems.

In cleanrooms, any system that affects these following real-time

parameters will need qualification: air purity (particle concen­tration/
ISO class), room temperature, humidity, airflow patterns, room
pressure, airflow direction between adjacent rooms, particle migrat­
ion direction between adjacent rooms, room enclosure integrity, etc.

Before a qualification plan is defined, a system’s critical and

non-critical parameters should be determined by means of a risk
analysis for all installation components, subsystems and controls.
Any parameter that may affect the quality of the pharmaceutical
product, or a direct impact component, should be considered a
critical parameter, and included in the qualification process. Non-
critical systems and components should be defined and may not
necessarily require qualification (FDA, 2004; WHO, 2007). A change
control procedure should be followed when changes are planned
 Cleanroom Commissioning and Qualification 293

to a system, its components and controls which may affect critical

parameters.

Qualification consist of four parts described as Design

Qualification (DQ), Installation Qualification (IQ), Operational
Qualification (OQ), and Performance Qualification (PQ), more
detailed discussions are in the following sections.

DESIGN QUALIFICATION
Design qualification is to provide the documented verification that
the proposed design of the facilities, systems and equipment is
“suitable for the intended purpose”. Early in design, it should be
determined who will be responsible for and how to produce as-built
drawings, maintenance files, and training. The design qualification
plan for the HVAC systems may include:

• Functional description of what the systems do along with

specific process and room requirements.

• Maps of room classification and pressurizations, airflow

diagrams, and cleanliness zones served by each air handling
unit.

• List of critical components to be qualified, including the

automation system controlling the HVAC.

• List of owner’s procedures that must be followed for qualifica-

tion of equipment and systems that affect critical parameters.

• List of qualification procedures (IQ/OQ/PQ protocols) written

especially for the project.

• List of equipment requiring commissioning, determined

through a risk-based product and process impact analysis.
294 Cleanroom Contamination Prevention and Control

It is important to measure and document critical variables of a

system (e.g., space pressure), but it is also important to document
and record performance requirements and results for components
that affect the critical parameters (e.g., room pressure sensors,
temperature sensors, airflow volume sensors) for GMP records.
Documentation helps ensure that replacement equipment/parts can
be specified, purchased, and installed to support critical operations.

It is important to determine all components and instruments that

could affect critical parameters and could, through an undetected
failure, lead to product adulteration. This may be accomplished by a
joint effort among the facility design engineer, owner, quality control
experts, and a qualified protocol writer. If performance data are in the
qualification records, replacement parts of different manufacturers
may be installed without major change control approvals, as long
as they meet performance requirements. Owner approval for the
qualification plan should be obtained during detailed design phase.

INSTALLATION QUALIFICATION
Installation qualification is to provide inspections to verify comp­
liance with contract documents, including completion of punch list
work, for critical components. It may include material test reports,
receipt verification forms, shop inspection reports, motor rotation
tests, duct/equipment cleaning reports, duct leak testing, piping
and instrumentation diagrams (P&ID) walkdowns for component
installation inspections, and contractor-furnished testing and
balancing. It also includes calibration records for instrumentation
used in commissioning and for installed instrumentation (e.g.,
sensors, recorders, transmitters, controllers, and actuators). The goal
is to ensure that the facility and process equipment are installed per
the contract documents.

Control software should be bench tested, and preliminary

tuning parameters should be entered for possible operational modes.
Control loops should be dry-loop checked to verify that subsystem
installation, addressing, operation, and graphics are correct.
 Cleanroom Commissioning and Qualification 295

Equipment and instruments should be tagged, wiring labeled, and

verified in field against record drawings. Updated commissioning
documentation must attest to completion of these activities and
include as-built drawings and installation/operation/maintenance
(IOM) manuals from contractors and vendors. The discussions
below focus more on the qualifications of utilities, equipment and
HVAC systems.

Utilities and equipment

Qualification of utilities and equipment is critical to demonstrate
and document compliance with all requirements, and generally
includes the following activities:

• Selecting utilities and equipment construction materials,

operating principles, and performance characteristics to ensure
that they are appropriate for their specific uses.

• Verifying that utility systems and equipment are built

as designed and installed in compliance with the design
specifications, with proper materials, capacity, and functions,
and properly connected and calibrated.

• Verifying that utility systems and equipment operate in

accordance with the process requirements in all anticipated
operating ranges. This should include challenging the
equipment or system functions while under load comparable to
that expected during routine production. It should also include
the performance of interventions, stoppage, and start-up as is
expected during routine production. Operating ranges should
be shown capable of being held as long as would be necessary
during routine production.

Before any batch from the process is commercially distributed for

consumers, a manufacturer should have a high degree of confidence
in the performance of the manufacturing process and that it will
consistently produce APIs and drug products meeting requirements
296 Cleanroom Contamination Prevention and Control

relating to identity, strength, quality, purity, and potency. Assur­

ance should be obtained from objective information and data
that demonstrates that the manufacturing process is capable of
consistently producing acceptable quality.

HVAC and environmental control systems

Qualification of the pharmaceutical cleanroom’s HVAC and environ­
mental control systems is part of the overall qualification of the
facility. Equipment affecting critical parameters and their control
must also be qualified. The most important objectives in meeting the
approving agency requirements are to (ASHRAE, 2019):

• State what procedures will be followed and verify that it was

done.
• Show that product is protected and acceptance criteria are met.

OPERATIONAL QUALIFICATION
Operational qualification is to ensure that the facility and process
equipment operate as intended throughout all anticipated ranges
of performance. This qualification will provide documentation for
start-up, operation, and maintenance, as well as ensure that Standard
Operating Procedures (SOPs) are correct and activated for critical
systems and components. This includes individual performance
testing of control loops under full operating conditions in a logical
order (i.e., room airflow control before room pressure control). The
commissioning agent must verify that operating parameters are
within acceptance criteria.

The HVAC system may be challenged under extremes of design

load to verify operation of alarms and recorders, to determine and
correct if significant weak points exist, and to verify control and door
interlocks. Based on observations, informal alert values of critical
parameters that might signify abnormal operation may be set up.
Even if the product would not be adulterated at these parameter
 Cleanroom Commissioning and Qualification 297

values, staff may implement an alarm to require responses prior to

encountering deviations from normal operation.

Documented smoke visualization tests can verify particle

migration between rooms, room airflow patterns in critical spaces or
inside containment equipment, as well as visualize airflow patterns
and dir­ ections around critical parts of production equipment.
Smoke tests should be videotaped as evidence for each operational
con­dition, especially when room airflow patterns are disrupted
or distorted, or room pressure differentials are lower than the
acceptance criteria. The videotaped evidence can help a subject
matter expert identify the root causes of the problems and provide
solutions to remediate and retrofit the systems as necessary.

Files should include an updated description of the HVAC,

describing how it operates, schematics, airflow diagrams, and the
space pressure maps that accompany these documents. Copies
should be readily accessible and properly filed. Operating person­
nel should be familiar with the data in these records and be able to
explain it to an agency inspector.

GMP documents should also include test reports for HEPA

filters (efficiency or pinhole-scan integrity tests) at final operating
velocities. To avoid conflict of interest, the qualification test should
not be performed by the filter installer, the data should be part of the
IQ package.

Documents should verify that instruments display, track, and

store critical parameters and action alarms. Consider recording
data by exception and routine documentation of data at a regular
frequency.

Systems and equipment should be entered into the owner’s

maintenance program, including rough drafts of associated mainte-
nance procedures (final drafts should reflect commissioning results).
298 Cleanroom Contamination Prevention and Control

Records should document the completion of these activities,

including final as-built, system diagrams, facility pressurization
diagrams, air change rate calculations, and air and water balance
reports.

PERFORMANCE QUALIFICATION
Performance qualification is to ensure that the facility and process
equipment perform as they are intended and meet predetermined
acceptance criteria over a specified period. This qualification is to
provide the proof that the systems perform as intended under actual
production conditions. PQ is the beginning of ongoing verification
(often called validation) that the system meets acceptance criteria
of the product. In medical product manufacturing microbiological
testing is as important as particle testing during performance quali-
fication. As microbial contamination from the cleanroom poses risk
to product as well as patient, this testing has to be extensive during
PQ. It may range from four to six weeks of testing to understand the
bioload in the critical and surrounding rooms and its effect on open
product during manufacturing. This includes documentation of:

• Maintenance record keeping and final operating and main­

tenance procedures in place, with recommended frequency
of maintenance, and (at the owner’s option) a procedure for
periodic challenge of controls and alarms.

• Logs of critical parameters that prove the system maintains

acceptance criteria over a prescribed amount of time.

• Training records for operators and maintenance personnel.

• Final loop tuning parameters.

After accepting PQ, the owner’s change control procedure should

limit further modifications to critical components (as shown on IQ
and OQ forms) that affect the product. Much of the facility’s HVAC
equipment should not need qualification, but records for the entire
facility must be kept up to date through quality change control, and
 Cleanroom Commissioning and Qualification 299

problems must be corrected before they become significant. Records

of corrections should also be kept.

Once the system is operational, pharmaceutical product trial

batches undergo process validation; the facility should regularly
monitor levels of viable (microbial) and nonviable particles, room
pressurization, and other controlled parameters in the processing
areas.

REQUALIFICATION
Periodic requalification of parameters should be executed at regular
intervals to demonstrate continued compliance. Requalification
should also be performed when any changes (such as changes to
utilities, systems, equipment, space configuration, maintenance
effort, or even personnel movement passage), which could affect
system performance, take place (WHO, 2007).

REFERENCES
ASHRAE (2019) “Clean spaces”, Chapter 19 in ASHRAE Handbook –
HVAC Applications. Atlanta: ASHRAE.

ASTM (2013) ASTM E2500-13. Standard guide for specification,

design, and verification of pharmaceutical and biopharma­
ceutical manufacturing systems and equipment. West
Conshohocken, PA: ASTM International.

FDA (2004) Guidance for industry: Sterile drug products produced

by aseptic processing – Current good manufacturing practice.
US Department of Health and Human Services, US Food and
Drug Administration, Silver Spring, MD.

IEST (2016) IEST-RP-CC001.6. HEPA and ULPA filters. Contamin­

ation Control Division Recommended Practice 001.6. Arlington
Heights, IL: Institute of Environmental Sciences and Technology.
300 Cleanroom Contamination Prevention and Control

IEST (2015) IEST-RP-CC012.3. Considerations in cleanroom design.

Contamination Control Division Recommended Practice 012.3.
Arlington Heights, IL: Institute of Environmental Sciences and
Technology.

ISO (2019) ISO 14644-3. Cleanrooms and associated controlled

environments – Part 3: Test methods. Geneva: International
Organization for Standardization.

ISPE (2001) Baseline Guide Volume 5: Commissioning and

Qualification.

NEBB (2009) Procedural standards for certified testing of cleanrooms.

3rd ed. Gaithersburg, MD: National Environmental Balancing
Bureau.

Sun, W. et al. (2017) ASHRAE Design Guide for Cleanrooms: Funda-

mentals, Systems, and Performance. Atlanta: ASHRAE.

WHO (2007) Quality assurance of pharmaceuticals – A compen­

dium of guidelines and related materials, Volume 2. Good
manufacturing practices and inspection. World Health
Organization.

ABOUT THE AUTHOR

Mr. Sun has been working in the US as an engineer for 28 years
in building mechanical systems design, HVAC, consulting, training
and research in his current position as the President at Engsysco, Inc.
Mr. Sun is a Fellow, distinguished lecturer, a member of the
nominating committee and chairman of the technology transfer
committee at the American Society of Heating, Refrigerating and Air
Conditioning Engineers (ASHRAE).
 Cleanroom Commissioning and Qualification 301

He was the chairman of TC 9.11 committee for clean spaces as

well as a member of TC 9.6 committee for healthcare facilities He
was also the President of the ASHRAE Detroit Chapter in 2010–2011.
As the principal author, his 450-page new technical book ASHRAE
Design Guide for Cleanrooms was published in 2017. He led over 20 co-
authors and spent six years in accomplishing this major achievement.

In other leadership roles, Mr. Sun served as the President of

Institute of Environmental Science and Technology (IEST) Society
in 2016–2017 in charge of all society leadership and management
responsibilities. He is also the USA Delegate for ISO cleanroom
standards 14644 on behalf of the USA since 2012. Mr. Sun is also the
chairman of the National Environmental Balancing Bureau (NEBB)
for the cleanroom performing testing standard (CPT).
 10

HUMAN BORNE CONTAMINATION –

WHEN CLEANROOM REUSABLE
OR DISPOSABLE GARMENTS
CANNOT BE A GOOD BARRIER

Jan Eudy
Jan E. Eudy Consulting
Carolina Beach, NC
USA

Reusable or disposable, single-use cleanroom garments are personal

protective equipment (PPE) for trained personnel working in
ISO Class 3-9/Grade A-D cleanrooms. The cleanroom garments
are designed to minimize human sourced contamination in the
cleanroom. However, if the garment selected doesn’t fit properly,
is constructed of inferior fabric or components with poor seam
construction, or inferior packaging or incorrectly laundered; then
the human sourced contamination is increased and this scenario will
compromise the cleanroom.

303
304 Cleanroom Contamination Prevention and Control

THE IMPORTANCE OF CORRECT SELECTION

OF REUSABLE OR DISPOSABLE, SINGLE-USE
CLEANROOM GARMENTS
Fabric
There are two types of recommended cleanroom fabrics; reusable,
woven fabrics and disposable, single-use non-woven fabrics. Each type
of fabric has specific advantages and disadvantages. Reusable, woven
cleanroom fabrics are manufactured using 100% continuous filament
polyester and continuous filament polyester/carbon combination
yarns to minimize particle shedding from the yarn. Reusable fabrics
chosen must be evaluated for cleanability using the recommended
cleanroom garment laundering process per IEST-RP-CC003, Garment
System Considerations for Cleanrooms and Other Controlled Environments
and compatible with a validated sterilization process. Since 1995,
the high performance, high density electrostatic dissipative (ESD)
cleanroom fabrics developed by Burlington Industries, Inc. and
Precision Fabrics Group, Inc. have been the preferred fabrics used
in reusable cleanroom garments. These fabrics have a plain taffeta
weave that is calendared to create a very small pore size of less than 10
micrometers which imparts improved bacterial and particle filtration.
Fabrics with a twill or herringbone weave have larger pores, have
more tendency to pill and are easily abraded causing contamination
of the cleanroom. The semiconductor, microelectronics, aerospace,
nanotechnology and paint spray cleanroom industries specify
a polyester/carbon grid cleanroom fabric whereas the medical
device, pharmaceutical, biopharmaceutical, nutraceutical and food
cleanroom industries specify a polyester/carbon stripe cleanroom
fabric. Beltron™ is the trade name of the carbon fiber used in the
Burlington Industries, Inc. and Precision Fabrics Group cleanroom
fabrics. The addition of the Beltron™ carbon yarn ensures that the
fabric is static dissipative which prevents unwanted particle migration
due to electro-inductive forces. Both companies have validation test
data on the fabrics recommended for use in cleanroom garments. The
Precision Fabrics Group, Inc. cleanroom fabrics also have a durable
Teflon™ and a broad-spectrum antimicrobial suffused in the weave
ensuring the fabric is liquid repellant and reduces bioburden.
 Human Borne Contamination 305

There are Asian alternatives of both the Burlington Industries

and Precision Fabrics Group cleanroom fabrics. The end user must
review fabric test data performed by third party testing laboratory
to assure quality, functionality and durability of these fabrics.

Nomex™ fabric is constructed of continuous filament DuPont

Nomex™ yarns. Only Nomex fabric and Nomex sewing thread is
used in the construction of arc thermal/arc flash reusable cleanroom
garments. These garments are cleanroom launderable and can be
gamma sterilized for use in cleanrooms for sterile products which may
be aseptically produced and terminally sterilized. These garments
should be considered in high risk low bioburden cleanrooms too.
However, more rigorous periodic testing of the cleanroom garment
integrity must be performed to assure the efficacy of the cleanroom
garment and the safety of the cleanroom operator. There are no
disposable, single-use non-woven fabrics used in the construction of
arc thermal/arc flash cleanroom garments.

Disposable, single-use non-woven fabrics are constructed of

polyolefin fibers. A polyolefin is any long-chain synthetic polymer
composed of at least 85% (by weight) of ethylene, propylene or other
olefin units. The fibers typically used in disposable, single-use non-
woven fabrics are:

• Polypropylene (a polyolefin).
• Polyethylene (a polyolefin).
• Polyester.

There are six types of disposable, single-use non-woven

cleanroom fabrics. Spunbonded fabrics are made from continuous
polypropylene filaments which increase strength and reduce
particulation but in an open structure. These fabrics do not have
high barrier performance and are used in the construction of
bouffants and shoe covers. DuPont Tyvek™ fabric is a flash spun
non-woven fabric made of high-density polyethylene continuous
fibers. Flash spun fabrics have barrier properties and are splash-
resistant. Because melt blown nonwoven fabric does not have
306 Cleanroom Contamination Prevention and Control

adequate strength to be used alone for disposable, single-use

cleanroom garments, spunbonded/melt blown/spunbonded (SMS)
is a laminate fabric that offers barrier properties and comfort. Film
laminate non-woven fabrics are a spunbonded layer laminated to a
nonporous film. This non-woven fabric demonstrates particle, blood
and chemical barrier characteristics. Microporous film laminate
disposable, single-use non-woven fabrics are a spunbonded layer
laminated to a microporous film. This non-woven fabric is splash-
resistant, blood and particle barrier. Disposable, single-use non-
woven fabrics can be sterilized and treated for antistatic properties.
All disposable, single use non-woven garments are designed to be
used only once and can withstand ionizing radiation sterilization
up to 50 kGy. Both reusable, woven and disposable, single-use non-
woven fabrics are the most expensive component of cleanroom
garments. Selection of the fabric should be based on the performance
test results of the fabric.

Performance testing of the fabric

There are several American Society for Testing and Materials
(ASTM) and American Association of Textile Colorists and Chemists
(AATCC) test methods used to evaluate the fabrics.

• The weight of the fabric determines its strength and durability;

however, a lighter fabric contributes to operator comfort (ASTM
D-3776). Operator comfort is extremely important for medical
product manufacturing.

• The pore size is an indicator of barrier efficiency. The smaller the

pore size, the more particles that will be entrained. Calendaring
is a process used on reusable, high performance, high density
ESD fabrics to reduce pore size (Porometer method).

• The moisture vapor transmission rate (MVTR) evaluates the

ability to remove moisture through the fabric and translates
to operator comfort. Most disposable, single-use non-woven
fabrics have low MVTR results (ASTM D96-80).
 Human Borne Contamination 307

• Particle filtration (ASTM F2101) and bacterial filtration efficiency

(ASTM F2299) tests for the ability of the fabric to contain both
viable and non-viable contaminants.

• Air permeability is the ability of a fabric to allow air to pass

through it. The lower the permeability of air within the fabric to
the outside, the lower the contamination to the product (ASTM
D737).

• Splash resistance or the ability of the fabric to be penetrated by

liquids or chemicals allow the cleanroom operator to be better
protected from spills in the cleanroom environment.

• Static decay (FTM-4046) and surface resistance (AATCC-76)

testing of the fabric is performed to assure the fabric is static
dissipative.

Seams
A seam is a joint consisting of a sequence of stitches uniting two or
more pieces of material. Prior to stitching any seams, the edges of
the cleanroom fabric panel should be properly prepared in order
to eliminate the possibility of loose threads, frayed edges or seam
separation which may cause contamination of the cleanroom.
Reusable, woven cleanroom fabric edges are either overlock (serged)
stitch or laser fused edge. Disposable, single-use non-woven
cleanroom fabrics are typically heat-sealed for a fused, beaded edge
using pressure and a heat-activated adhesive. This step is often not
performed because the cleanroom garment manufacturer wants to
reduce the cost of manufacturing the cleanroom garment and the
end user won’t notice until the garment is causing a contamination
problem in the cleanroom.

The two types of joining seams recommended for the

construction of cleanroom garments are bound with single needle
stitching and lapped (flat feld) with double needle stitching. ASTM
D-6193-16, Standard Practice for Stitches and Seams stipulate seams
308 Cleanroom Contamination Prevention and Control

used in construction of personnel protective garments should

contain 6–7 stitches per inch. Entrapment areas are stitched features
of the cleanroom garment that can trap unwanted contaminants.
Unnecessary seams and features should be avoided. Pockets, badge
or pen tabs, ear plackets and beeper slots are not recommended as
additions to cleanroom garments. Serged seams that are not bound
or lapped are not recommended for any cleanroom garment. Many
cleanroom frocks and undergarments that are worn in ISO Class 7
and 8 cleanrooms are sewn with only serged seams. These seams
continually shed particles and fibers. The seams are more durable
if the number of stitches per inch is at minimum 6–7 stitches per
inch. The lesser the number of stitches per inch, the more potential
break-thru of particles through the seams. All disposable, single-use
cleanroom garments should have bound seams. Often contractors
will be working in a cleanroom with lesser quality disposable,
single-use garments with serged seams. This can cause unnecessary
contamination in the cleanroom.

Components
Components or “findings” are supplementary items used in the
construction of both reusable, woven and disposable, single-use non-
woven cleanroom garments. They should be cleanroom compatible
and gamma irradiation compatible. They should be installed on the
garment in a way that minimizes the entrapment and/or release of
contamination while in use.

• Snaps should be post and socket type stainless steel or plastic.

Lesser grade stainless steel is not compatible with multi-
megohm deionized (DI) water rinses used as the final rinse
in the cleanroom laundering cycle for reusable cleanroom
garments. Lesser grade stainless steel snaps will corrode and
cause particulation when the garment is worn in the cleanroom.

• Binding tape and straps should be of the same reusable, woven

or disposable, single-use non-woven cleanroom fabric as the
cleanroom garment.
 Human Borne Contamination 309

• Only polyester, silicone free thread must be used in the

construction of all cleanroom garments. Silicone and other
similar organic compounds are airborne molecular contaminants
(AMC) which will cause a film deposition in optic cleanroom
manufacturing, “fish eyes” in paint spray cleanroom operations
and various contamination in defense cleanroom manufacturing.
Polycotton blend thread should never be used to sew cleanroom
garments. Cotton is particulating and not gamma compatible.

• Polyester zippers are the recommended closures for cleanroom

coveralls and frocks. Hook and loop closures such as Velcro®
are not recommended because they pose a significant risk of
trapping and releasing contaminants in the cleanroom.

Garment style
The garment style recommended for both reusable, woven and
disposable, single-use non-woven cleanroom garments utilizes a
minimal number of seams and components.

• Coveralls are a one-piece garment with a zipper closure covering

the entire body from the neck to the wrists and ankles. Coveralls
with a zipper covering (placket) meet CE 0321 Type 5 and
Type 6 specifications for both reusable and disposable, single-
use cleanroom garments used in Europe. The diameter of the
leg should be large enough for typical shoes worn in cleanrooms
to slide easily through the opening without snagging or tearing
and fit comfortably into the legging of the cleanroom boot. A
series of snaps below the knee level can be used to secure the
boot to each leg of the coverall.

• Both reusable and disposable, single-use cleanroom boots are

a covering for the foot and lower portion of the leg. The sole
material is typically non-skid for wearer safety and compatible
with the application environment. The upper portion of the boot
should be high enough to cover pant leg up to the calf. Snaps,
ties and elastic at the top of the boot secures the boot to the leg
310 Cleanroom Contamination Prevention and Control

of the coverall. The sizing and fit of the boot should be snug
over the shoe to prevent slips, trips or falls in the cleanroom.
Adjustable straps will secure the boot over the shoe.

• Shoe covers are designed to contain contamination and should

cover the shoe. Reusable cleanroom fabrics are typically not as
effective as the disposable, single-use shoe cover. The sole should
be non-skid. Some shoe covers have a carbon grounding strip
that is placed inside the wearer’s street shoe. It is recommended
if street shoes are worn into the cleanroom gowning room that
they are cleaned by stepping on a sticky mat or contamination
control flooring and covered with a disposable, single-use shoe
cover prior to entering the gown room and donning boots.

• Bouffants are light-weight and elasticized hair covers. Both

reusable woven and disposable, single-use non-woven bouffants
must completely cover all hair and ears. Bouffants are typically
donned before entering the gown room whether or not they are
worn under hoods. Loose weave hair nets are not recommended
as they will allow contamination (particles, dandruff and
microbes) to escape and inadvertently carried into cleanroom.

• Hoods are coverings designed to fit over the head and generally
cover all but the facial area. Hoods should cover the head, neck,
shoulder and facial area (including forehead) to contain and
control migration of hair and particles. The ties, snaps, elastic
or a combination of all at the back of the hood enables the hood
to be fitted properly to provide a snug fit to the wearer’s head.
Reusable hoods may have sewn-in facemasks or snaps to insert
reusable or disposable facemasks. Disposable, single-use hoods
typically are a pullover, full face opening design with a full
drape over the shoulders. A split bib drape on the reusable hood
is not recommended due to the increased likelihood of a particle
excursion at the neck area when moving the head. The hood
should be constructed of the same fabric as the coverall and boot
to limit human sourced contamination.
 Human Borne Contamination 311

• Frocks are three-quarter length garments with a military collar

design similar to cleanroom coveralls and a full front closure
with snap adjustment at the neck opening. Frocks are used in
cleanrooms of less critical air cleanliness classifications. A full-
length zipper closure is recommended rather than snap front
closure to prevent contamination generated by the wearer’s
street clothing from escaping into the cleanroom area. Frocks
should not have pockets and other entrapment areas.

• Gloves must be cleanroom compatible and powder free. Gloves

may be reusable, polyester or disposable vinyl, PVC, latex or
nitrile. In order to be cleanroom compatible, all gloves must be
packaged in plastic bags; not cardboard boxes. For some clean-
room industries, there are banned substances such as silicone,
amide and Dioctyl Phthalate (DOP) that glove manufacturers
have to avoid. All cleanroom industries require consideration
in the particle count, nonvolatile residue and ionic residue on
the surface of the gloves. For more detailed information on the
selection of gloves refer to IEST-RP-CC005, Gloves, Finger Cots
used in Cleanrooms and Other Controlled Environments.

• The goggles, prescription glasses or safety glasses must be

frequently cleaned during use and when initially put on in
the gown room. 70% isopropyl alcohol, preferably sterile, is
recommended for cleaning non-sterile goggles, prescription
glasses or safety glasses. For aseptic manufacturing, regulators
prefer using sterile goggles, not those sanitized using 70% IPA.

THE CLEANROOM LAUNDERING PROCESS

• The cleanroom garment laundry process for reusable cleanroom
garments is clearly defined in IEST-RP-CC003, Garment
Considerations for Cleanrooms and Other Controlled Environments.

• The soiled garments are retrieved from the cleanroom customer

and transported to the cleanroom garment laundry facility. The
cleanroom garment laundry will sort the garments by type and
312 Cleanroom Contamination Prevention and Control

color and scan the garment if bar coded. The initial inspection
will identify garments that are unserviceable due to rips, tears
or stains. These garments are segregated and stain treated,
repaired or replaced.

• The cleanroom garments are laundered in a programmable,

stainless steel pass thru washer. The soiled garments are loaded
on the soiled side of the washer and unloaded in a certified
cleanroom environment. The wash process consists of using
filtered softened or reverse osmosis (RO) treated water, in
conjunction with filtered non-ionic surfactants (detergents). The
final rinse water quality is multi-megohm (15–18 MΩ) deionized
water filtered to equal to or less than 0.22 microns. The wash
temperature is approximately 60°C or 140°F and gradually
reduced to room temperature using multiple rinses. The wash
temperature is critical to garment durability.

• The specially fitted cleanroom dryers have incoming air passed

thru high-efficiency particulate air (HEPA) filters in one pass at
an air temperature of less than 60°C or 140°F. The washers and
dryers are typically loaded at less than 75% capacity to assure
thorough washing and drying of the cleanroom garments.

• After drying, the cleanroom garments are inspected and folded.

The garments are inserted into cleanroom compatible and
gamma compatible packaging that is certified to IEST-STD-1248
cleanliness. The garments are folded in such a fashion as to
be easily removed from the package and donned in the gown
room with minimal difficulty in the gowning process. The final
packaging for shipment or delivery is based on the customer’s
requirements.

• The cleanroom garment laundries should have a robust quality

assurance program that regularly tests the garments using
the Helmke Tumble Drum test method for compliance to
IEST-RP-CC003 Category 1 particle cleanliness. A Certificate
of Conformance typically accompanies the shipment of lot
 Human Borne Contamination 313

of garments tested as well as other documents detailing lot

traceability, packing list or other quality assurance certifications.
Periodic testing for garment integrity should be performed
to assure the guaranteed useful life of the garments and
documented in the tracking system.

Figure 1 Helmke garment cleanliness classification

IEST-RP-CC003.3 Cleanliness Classification Chart

Category Garment Type Particle emission rate, G particles/min
0.3µm and larger 0.5µm and larger
I 1 Frock Less than 1,700 Less than 1,000
I 1 Coverall Less than 2,000 Less than 1,200
I 3 Hoods Less than 780 Less than 450
II 1 Frock 1,700 to 17,000 1,000 to 10,000
II 1 Coverall 2,000 to 20,000 1,200 to 12,000
II 3 Hoods 780 to 7,800 450 to 4,500
III 1 Frock 17,000 to 170,000 10,000 to 100,000
III 1 Coverall 20,000 to 200,000 12,000 to 120,000
III 3 Hoods 7,800 to 78,000 4,500 to 45,000
Note: The particle emission rates shown for each of the garments types are
proportional to the respective areas of fabric involved. The areas of the garments
considered in the preparation of the table are as follows:
Garment Average area, Average area, ft² (both sides)
Type m² (both sides)
Frock* 4.63 49.8
Coverall* 5.99 64.4
Hood* 1.03 11.0
*Medium size garments

STERILE CLEANROOM GARMENTS

• Gamma irradiation sterilization is the most common method of
sterilization of cleanroom apparel. Gamma irradiation sterilizes
the garments using Cobalt 60 ionizing energy. This method has
a high penetration of materials and lethal effects on microbial
populations at very low levels of radiation. Gamma radiation
314 Cleanroom Contamination Prevention and Control

sterilization is a reproducible and reliable process that is based on

the exposure time of the product to the gamma source. Regardless
of the sterilization method, gamma or e-beam radiation,
all cleanroom materials, including reusable and disposable
cleanroom garments, used in sterile cleanroom manufacturing
must undergo a validated dose audit test for sterility assurance
levels using the following standards as applicable.

• ANSI/AAMI/ISO 11137-1: 2006 Sterilization of Health Care

Products – Requirements for Development, Validation and
Routine Control of a Sterilization Process for Medical Devices.

• ANSI/AAMI/ISO 11137-2: 2013 Sterilization of Health Care

Products – Radiation – Part 2: Establishing the Sterilization Dose.

• ANSI/AAMI/ISO 11137-3: 2006/(R) 2010 Sterilization of health

care products – Radiation – Part 3: Guidance on dosimetric
aspects.

• ANSI/AAMI/ISO 11737-1: 2006(R) 2011 Sterilization of healthcare

products – Microbiological methods – Part 1: Determination of
the population of microorganisms on product.

• ANSI/AAMI/ISO 11737-2: 2009 Sterilization of medical devices

– microbiological methods – Part 2: Tests of sterility performed
in the definition, validation and maintenance of a sterilization
process.

• ANSI/AAMI/ISO TIR 13004: 2013 Sterilization of healthcare

products – Radiation – Substantiation of a selected sterilization
dose: Method VDmaxSD.

• Cleanroom apparel may be sterilized using ethylene oxide

(ETO). ETO sterilization is sometimes used for disposable
cleanroom garments. These garments must be segregated for
two weeks to “outgas” after the sterilization process. Sterility
validation is performed using ANSI/AAMI/ISO 11135-1: 2007,
 Human Borne Contamination 315

Sterilization of health care products – Ethylene oxide – Part 1:

Requirements for development, validation and routine control
of a sterilization process for medical devices.

• Cleanroom apparel used in the United Kingdom, Europe and

Asia may also be steam sterilized. However, steam sterilization
of cleanroom apparel causes shrinkage and wrinkling of the
reusable garment system. Some manufacturers with in-house
laundries iron the laundered garments to remove wrinkles which
further compromises garment integrity. Additionally, one must
certify that the cleanroom compatible components used in the
cleanroom garments are not destroyed by steam sterilization.
Sterility validation of steam sterilization is performed per ISO/
TS 17665‑3: 2013, Sterilization of health care products – Moist
heat – Part 3: Guidance on the designation of a medical device to
a product family and processing category for steam sterilization.

• Historically, regulated medical product manufacturers that are

producing a product in an aseptic cleanroom with a final product
sterility assurance level (SAL) of 10-6 will specify that all materials
required during the manufacturing process be validated and
certified to 10-6 SAL. However, FDA regulated, terminally steril­
ized, non-implantable medical device manufacturers in a non-
sterile cleanroom may specify that all cleanroom materials used
in the process be validated and certified sterility to 10-3 SAL.

• It is in the best interest of the cleanroom materials manufacturer

to reduce the average device bioburden levels during the
manufacturing process to in turn reduce the level of gamma
radiation required to terminally sterilize the product to the
sterility assurance level required by the customer. It is also most
cost effective to radiate the manufactured materials to the same
sterility assurance level. Therefore, most suppliers of sterile
cleanroom materials validate their processes to deliver sterile
cleanroom reusable or disposable materials at 10-6 SAL to serve a
broader, universal market. This validation consists of performing
quarterly dose audits. A dose audit consists of performing
316 Cleanroom Contamination Prevention and Control

a bioburden analysis of the medical device, i.e., cleanroom

garment sample item portion (SIP). Once the average device
bioburden is determined, the verification dose is established
and the required number of devices (i.e., cleanroom garment
SIP) are radiated. After radiation, the samples are evaluated
for sterility. During the bioburden analysis, the three most
dominant microorganisms are identified macroscopically and
microscopically by performance of a Gram stain. If there are
positive samples after sterility testing, these microorganisms are
identified to genus and species.

• One must also validate the packaging of the cleanroom apparel,

determine the maximum tolerated dose of gamma radiation and
test for strength, discoloration and durability. Additional shelf-
life testing is performed to assure sterility for an extended period
of time – usually one calendar year for cleanroom reusable
garments and up to five years for cleanroom disposable, single-
use garments.

GAMMA SUBCONTRACTOR QUALIFICATION

The gamma subcontractor must be audited and approved by the
cleanroom apparel provider. This is usually an on-site audit that
verifies the equipment validation and dose mapping performed by
the gamma irradiator to determine sites of minimum and maximum
dose ranges. The dose is variable with respect to the density of the
packaging, therefore it is imperative that the density remain uniform
in the mapped product.

ROUTINE MONITORING OF GAMMA RADIATION

Colorimetric indicators may be used as a visual aid to show that the
garments have been exposed to irradiation. Indicators for gamma
radiation change from yellow to red and are typically placed on
inner and outer packaging. If an indicator is exposed to IPA such
as during decontamination of the external surface of the packaging
 Human Borne Contamination 317

(wiping) prior to placing the garment item in the sterile gowning

area, the IPA can cause the red indicator (one that has been exposed
to irradiation) to turn back to yellow.

CERTIFICATE OF STERILITY
Each lot of garments gamma processed will be issued a certificate
of sterility based on the evaluation of the dosimetry data results of
the irradiation run. A typical certificate of sterility will contain the
following information:

• Product description or catalog number.

• Lot number.
• Irradiation run number.
• Date of irradiation.
• Statement of how the product was sterilized and how the process
was validated.
• Minimum and maximum specified dose.
• Minimum and maximum delivered dose.
• Signature, date and title of individual performing dosimetry
testing.
• Signature, date and title of individual performing review of test
data.

It is imperative that the complete supply chain of the disposable and/

or reusable cleanroom garments is audited for compliance to IEST-
RP-CC003.4 and to individual customer specifications. Reusable
cleanroom garments should have established evaluation protocols
over the life cycle of the garment and beyond use criteria for both
sterile and non-sterile cleanroom garments. Routine scheduled
assessments assure that the cleanroom garments protect the process
and the product in the cleanroom.
318 Cleanroom Contamination Prevention and Control
Figure 2 Recommended garment system configurations
applicable to various cleanrooms

Apparel ISO ISO ISO ISO ISO Class ISO ISO ISO Class
Type Class 8 Class 7 Class 6 Class 5 for Class 4 Class 3 1 and ISO
5 Aseptic Class 2
Inner Suit AS AS AS R AS R R R
Hair Cover R R R R R R R AS
(Bouffant)
Woven AS AS AS AS NR NR NR NR
Gloves
Barrier AS AS AS AS R R R R
Gloves
Facial AS AS AS R R R R AS
Cover
Hood AS AS AS R R R R AS
Powered AS AS AS AS AS AS AS R
Headgear
Frock R R AS AS NR NR NR NR
Coverall AS AS R R R R R R
Two-Piece AS AS AS AS NR NR NR NR
Suit
Shoe Cover R R AS AS NR NR NR NR
Boot AS AS R R R R R R
Special AS AS AS AS AS AS AS AS
Footwear
Suggested 2x/week 2x/week 3x/week 1x/day Per Entry Per Per Per Entry
Frequency Entry Entry
Change*
* Consideration should be given to the effects of seasonal conditions in some geographical areas.
Note that the suggestions made are not based on scientific data but instead reflect the collective
experience offered by the working group. Individual requirements for change frequency should be
determined on a case-by-case basis.
R = Recommended NR = Not Recommended AS = Application Specific

The most important, universal specification for cleanroom

disposable and reusable apparel is that the cleanroom garment
system is appropriate to protect the cleanroom process or product
manufactured in the cleanroom and that it is always worn correctly.
Refer to IEST-RP-CC003.4 Gowning configurations and change frequency
 Human Borne Contamination 319

for the recommended garment system and change frequency for

different Class cleanrooms.

APPLICATIONS SPECIFICALLY FOR DISPOSABLE

SINGLE-USE CLEANROOM GARMENTS
Hazardous materials
Disposable, single-use cleanroom garments are worn as personnel
protective equipment in cleanroom operations using hazardous
chemicals and cytotoxic drugs. In selecting the disposable, single-
use cleanroom garment system, the protective properties must be
evaluated. A third party ISO 17025 certified testing laboratory should
be used to test the disposable, single-use cleanroom garments for
liquid penetration and resistance to permeation by chemotherapy
drugs using methods described in ASTM F739 Standard Test Method
for Permeation of Liquids and Gases Under Conditions of Continuous
Contact and ASTM D6978 Standard Practice for Assessment of Resistance
of Medical Gloves to Permeation by Chemotherapy Drugs. Compounding
pharmacies will require permeation test results on:
• Carmustine.
• Cisplatin.
• Cyclophosphamide.
• Cyclosporin A.
• Doxorubicin Hydrochloride.
• Etoposide.
• Flourouracil.
• Methotrexate.
• Mitomycin C.
• Paclitaxel.
• Thiotepa.
320 Cleanroom Contamination Prevention and Control

Heavy soiling of fabric

Cleanroom garment laundries will charge extra fees for heavy soil
removal on reusable cleanroom garments. Disposable, single-use
cleanroom garments are recommended for cleanroom operations
that generate heavy soil on the fabric of the cleanroom garments.
Some disinfectants used in aseptic cleanroom operations will
permanently stain reusable cleanroom garments. Disposable, single-
use cleanroom garments that are liquid repellant are recommended
in cleaning and disinfecting aseptic cleanrooms.

Low volume usage

Some research operations access the cleanroom infrequently.
Disposable, single-use cleanroom garments are recommended
for cleanroom operations with low volume useage. Cleanroom
consultants and contractors also provide their personal disposable,
single-use cleanroom garments when providing their services to
cleanroom operations.

Case studies (Warning Letters 483s)

The following are observations regarding cleanroom garments
during regulatory audits generating Warning Letters:

• Protective apparel is not worn as necessary to protect drug products

from contamination. Specifically, your firm’s sterile technician’s
garment consists of non-sterile booties, non-sterile hair net, non-
sterile mask, sterile gowns and sterile gloves when cleaning the
ISO LAFH used to produce sterile injectable products. Your
firm’s current gowning practices leave the following eye and
neck areas exposed in the ISO 7 cleanroom containing the ISO
5 LAFH. I observed your sterile technician place their head
containing non-sterile hair net and non-sterile mask exposing
forehead, eye area and neck in the ISO 5 LAFH while cleaning
the ISO 5 LAFH prior to production.
 Human Borne Contamination 321

Author’s note: When working in a sterile (aseptic) ISO 5

cleanroom environment, it is recommended that sterile coveralls,
boots, hoods, masks, goggles and gloves are worn to prevent
contamination of the sterile cleanroom product.

• Clothing of personnel engaged in the processing of drug products is

not appropriate for the duties they perform. (a) Sterile drug products
are aseptically manipulated by clean room operators who
were observed wearing non-sterile gowns, non-sterile glasses/
goggles, non-sterile footwear, facial masks and non-sterile
bonnets. (b) The operator’s face and neck are not fully covered
allowing exposed facial skin and hair over that critical ISO 5
laminar flow areas where sterile injectable drug products are
manufactured.
Author’s note: When working in a sterile (aseptic) ISO 5 clean­
room environment, it is recommended that sterile coveralls,
boots, hoods, masks, goggles and gloves are worn to prevent
contamination of the sterile cleanroom product.

• Clothing of personnel engaged in the manufacturing, processing,

packing and holding of drug products is not appropriate for the duties
they perform. We observed a contract cleaning employee brush
the sleeves of her sterile gown (edges of the sleeves on the
jumpsuit) on the floor during the gowning procedure in gown
room prior to aseptic facility cleaning procedures.
Author’s note: If a cleanroom garment touches the floor while
donning the garment, it becomes contaminated with soil on the
floor. Remove the cleanroom garment and replace with a new
packaged cleanroom garment. Cleanroom garments should be
selected in the correct size for easy donning.

• Your cleanroom gowning procedure potentially compromises the

integrity of the sterile gowns used to form a barrier between the clean-
room operators and the area where sterile drug products are aseptically
manipulated in HEPA filtered laminar flow hoods. Your operators
wash and sanitize hands but then handle the outside of the
322 Cleanroom Contamination Prevention and Control

gowns (and sleeves) with their bare hands. Then they don a pair
of sterile gloves.
Author’s note: When donning sterile cleanroom garments, sterile
gloves are donned first and garment item. Cleanroom garments
should be folded and packaged for easy donning.

• Sterile gowning components are reused by employees on a daily

basis. Employees remove their sterile gowning components
(hanging them in ISO 7 classified room), leave and return from
lunch, and then re-gown for the afternoon work period, using
the same gowning components from the morning work period.
Author’s note: All sterile gowning components must be doffed
and discarded after each use. Sterile donning of sterile gowning
components is required upon each entry of the aseptic cleanroom
areas.

• Your firm failed to ensure that manufacturing personnel wear

clothing appropriate to protect drug product from contamination.
The garbing consisting of face masks, hair nets, gloves and suits
without hoods used at your facility is not adequate to protect
the drug product from microbiological contamination. During
your demonstration of cleaning and disinfection practices for
your aseptic processing room, our investigators observed an
operator who wore eye makeup with no eye protection. The
lack of the use of hoods allowed for exposed hair and skin on
their faces and necks. Furthermore, personnel reused these
suits on multiple aseptic processing days with no cleaning or
sterilization between use.
Author’s note: Sterile garments required for aseptic cleanrooms
are sterile coveralls, hoods, boots, mask, gloves and goggles
encapsulating the operator and covering the entire body.
Makeup is not allowed in any cleanroom operation. However,
I’ve observed some cleanroom operators with tattooed makeup
working in cleanroom operations. A tattoo requires a minimum
of one month to heal before the operator can work in a cleanroom.
All sterile gowning components must be doffed and discarded
 Human Borne Contamination 323

after each use. Sterile donning of sterile cleanroom garments is

upon each entry of the aseptic cleanroom areas.

• Poor aseptic behavior. An operator sitting on the cleanroom

floor during set-up of the filling line and not changing gown.
Operators leaning against cleanroom walls.
Author’s note: If the sterile cleanroom garments touch the floor or
walls at any time, they must be removed and discarded and new
sterile cleanroom garments must be donned.

• Your aseptic processing gowns were inadequate to prevent contamina-

tion of your sterile products with particles and microorganisms shed
from employees’ bodies. Our investigator observed employees
working in gowns that had unraveled stitching extending from
hoods, zippers and pants. Your firm approved these gowns for
operations. Employees wore them while manufacturing sterile
USP API and sterile API (Active Pharmaceutical Ingredients).
Five of 10 garments released for use in aseptic production areas
had loose fibers or other damage.
Author’s note: When selecting cleanroom garment suppliers,
include the Quality department in the initial supplier selection
and all ongoing qualification decisions. When selecting
reusable cleanroom garments, reduce the maximum number of
sterilization cycles to ensure reusable cleanroom garments are
replaced before the show signs of breakdown. When inspecting
sterile cleanroom garments prior to use, ensure that the Quality
department makes the final decisions relating to release of the
garments for aseptic production.

REFERENCES
AATCC 76 Electrical Surface Resistivity of Fabrics.

American National Standard, Association for the Advancement of

Medical Instrumentation (2016) AAMI TIR 35: 2016 Sterilization
of health care products – Radiation sterilization – Product
324 Cleanroom Contamination Prevention and Control

adoption and alternative sampling plans for verification dose

experiments and sterilization dose audits.

American National Standard, Association for the Advancement

of Medical Instrumentation (2013) ANSI/AAMI/ISO 11137-2:
2013 Sterilization of health care products – Radiation – Part 2:
Establishing the sterilization dose.

American National Standard, Association for the Advancement of

Medical Instrumentation (2009) ANSI/AAMI/ISO 11737-2: 2009
Sterilization of health care products – Microbiological methods
– Part 2: Tests of sterility performed in the definition, validation
and maintenance of a sterilization process.

American National Standard, Association for the Advancement of

Medical Instrumentation (2006) ANSI/AAMI/ISO 11737-1: 2006/
(R) 2011, Sterilization of health care products – Microbiological
methods – Part 1: Determination of the population of
microorganisms on product.

American National Standard, Association for the Advancement

of Medical Instrumentation (2006) ANSI/AAMI/ISO 11137-1:
2006 Sterilization of health care product – Radiation – Part 1:
Requirements for development, validation, and routine control
of a sterilization process for medical devices.

American National Standard, Association for the Advancement of

Medical Instrumentation (2006) ANSI/AAMI/ISO 11607-1: 2006
(R) 2015, Packaging for Terminally Sterilized Medical Devices –
Part 1: Requirements for materials, sterile barrier systems and
packaging systems.

American National Standard, Association for the Advancement

of Medical Instrumentation (2003) ANSI/AAMI ST 67: 2003
Sterilization of health care products – Requirements for products
labelled “STERILE”.
 Human Borne Contamination 325

American Society for Testing and Materials, ASTM D4169, Standard

Practice for Performance Testing of Shipping Containers and
Systems.

American Society for Testing and Materials, ASTM D7386, Standard

Practice for Performance Testing of Packages for Single Parcel
Delivery Systems.

Ask Jan (2007) Understanding Cleanroom Apparel Sterilization,

Part 1. Controlled Environments Magazine May. pp. 25–27.

Ask Jan (2007) Understanding Cleanroom Apparel Sterilization,

Part 2, Controlled Environments Magazine July/August pp. 34–35.

Association for the Advancement of Medical Instrumentation

(2005) AAMI TIR 33: 2005 Sterilization of health care products –
Radiation – Substantiation of a selected dose – Method VDmax.

ASTM D3776M-09a (2017) Weight of fabric. Standard Methods for

Mass per Unit Area.

ASTM F2299M-03 (2017) Standard Test Method for Determining the

Initial Efficiency of Materials Used in Medical Face Masks to
Penetration by Particulates Using Latex Spheres.

ASTM 96-80 Water Transmission Rate.

ASTM D737 Standard Test Method for Air Permeability of Textile

Fabrics.

ASTM F2101 Standard Test Method for Evaluating the Bacterial

Filtration Efficiency (BFE) of Medical Face Mask Materials Using
a Biological Aerosol of Staphlococcus aureus.
ASTM E 1294-89 F316 Pore size characteristics of membrane filters
using automated liquid porosimeter.

ASTM D-6193-16 Standard Practice for Stitches and Seams.

326 Cleanroom Contamination Prevention and Control

ASTM F739 Standard Test Method for Permeation of Liquids and

Gases Under Conditions of Continuous Contact.

ASTM D6978 Standard Practice for Assessment of Resistance of

Medical Gloves to Permeation by Chemotherapy Drugs.

FTM-4046 Electrostatic Decay.

Hazardous drug test results for Performance Qualification of

Lakeland CleanMax™ disposable, single use cleanroom
garments – March 14, 2019.

IEST-RP-CC003, Garment System Considerations for Cleanrooms

and Other Controlled Environments.

IEST-RP-CC005, Gloves, Finger Cots used in Cleanrooms and Other

Controlled Environments.

International Safe Transit Association, ISTA 3A, General Simulation

Performance Tests.

Madsen, R.E., Moldenhauer, J. (Eds.) (2018) Contamination Control in

Healthcare Product Manufacturing. Volume 5. Chapter 7, Gamma
Sterilization Validation of Disposable, Single-use and Reusable
Cleanroom Garments. PDA/DHI, Bethesda, MD. pp. 221–240.

Moldenhauer, J. (Ed.) (2015) Environmental Monitoring – A

Comprehensive Handbook. Volume 7. Chapter 3, Cleanroom
Gowning and Gowning Certification. PDA/DHI, Bethesda, MD.
pp. 43–63.
 Human Borne Contamination 327

ABOUT THE AUTHOR

Jan Eudy BSMT(ASCP)SM, RM(NRCM), CQA(ASQ) is a cleanroom
and contamination control consultant at Jan E. Eudy Consulting. She
has over 30 years cleanroom industry experience. She is a Fellow,
President-Emeritus and Senior Faculty member of the Institute for
Environmental Sciences and Technology and teaches courses for
the IEST.
 11

VIABLE AND NON-VIABLE

AIRBORNE PARTICLE MONITORING
EQUIPMENT SELECTION

Bill Shade
Microrite, Inc.
San Jose, CA
USA

OVERVIEW
Viable and non-viable particle monitoring are critical components in
reducing the risk of product contamination in sterile and non-sterile
pharmaceuticals as well as medical device manufacturing.

Particle monitoring is an extremely valuable tool for character-

izing and optimizing various contamination control aspects in all
cleanrooms and controlled environments.

Often only considered valuable in sterile/aseptic manufacturing,

viable and non-viable particle monitoring in equally important
for terminally sterilized, non-sterile and low bioburden products.
The risk of objectionable organisms is critical in all products as it
is directly related to patient safety. Whether the medicinal product
is sterile or non-sterile, viable and non-viable monitoring is a good
indicator of patient risk due to particle or microbial contamination.

329
330 Cleanroom Contamination Prevention and Control

Human borne organisms are considered the predominant

contributors of contamination in cleanrooms. Typically, 80 to 90
percent of normal microbial-flora identified in a cleanroom environ­
ment is generated from humans (Salaman-Byron, 2018). Adequate
gowning is the most effective way of protecting the cleanroom
environment from human borne contamination. Surfaces are
another potential source of contamination and are cleaned and
monitored on a regular basis for viable counts to rule out any foot
or wheel borne contamination. Finally, the air in the cleanroom is
a possible source of particles and particle levels are controlled by
high efficiency particulate air (HEPA) filters that have the capability
to filter out particles down to 0.2 µm in size. The air in the cleanroom
is controlled and monitored for particle counts and viable counts on
a regular basis; the frequency is dictated by the type of product and
the controls required.

A viable particle is a particle that contains one or more

living organisms. These can affect the microbial quality of the
pharmaceutical product and generally range from approximately 0.2
µm to 30 µm in size (Kelly, 2005). The method of monitoring these
particles is by capturing, incubating, counting and characterizing
them. Since it takes days to get results after incubating the samples,
viable particles are not counted in real time using conventional
microbiological methods. This time gap between when the sample
is taken during manufacturing and when the results are available
may increase the risk of a contamination in the product.

A non-viable particle is a particle that does not contain a living

microorganism but may act as transportation vehicle for viable
particles. They are measured in real time using optical particle
counters originally developed in the 1960s. These instruments
sample an air stream using vacuum pump technology and detect
individual particles reporting particle concentrations in the air at a
number of particle sizes throughout the size range of the instrument.
Because they are real time, they can be used to continuously monitor
critical locations in aseptic processing to reduce the risk of product
exposure to contamination going undetected. In addition, these
 Airborne Particle Monitoring Equipment Selection 331

instruments are used to certify, validate and routinely monitor

cleanrooms in accordance with industry standards to ensure the
room is providing clean air to the critical locations as required.

Because traditional viable particle monitoring is only capable

of detecting those particles that affect sterility or bioburden over
the course of days, and non-viable optical particle counters are
capable of detecting all contamination in the form of particles
in real time, an environmental monitoring plan requires both
techniques to minimize the risk of particles affecting the sterility
of, or compromising the manufactured medicinal product. Periodic
classification of the cleanroom in conjunction with frequent
monitoring based on a risk assessment is required by current
industry regulations and guidance.

INDUSTRY STANDARDS AND GUIDELINES

FOR NON-VIABLE PARTICLE MONITORING
All current Good Manufacturing Practices (cGMP) related
regulations and guidelines, including Food and Drug Adminis­
tration’s (FDA) sterile aseptic guideline, European Union (EU)
GMP, World Health Organisation (WHO), and PIC/S reference
International Cleanroom Standards (ISO 14644 series). The
classification of cleanrooms and controlled environments is
dictated in ISO 14644 Cleanrooms and associated controlled
environments, Part 1: Classification of air cleanliness by particle
concentration. Monitoring of cleanrooms is addressed in Part 2:
Monitoring to provide evidence of cleanroom performance related
to air cleanliness by particle concentration. Both ISO 14644 Part
1 and Part 2 were updated in 2015 and should be referenced as
following: ISO 14644-1: 2015 and ISO 14644-2:2015 respectively.

Cleanroom classification testing per ISO 14644-1 is performed

using light scattering optical particle counters in order to count
non-viable particles. ISO 14644-1 specifies classes of air cleanliness
for the world’s cleanrooms and controlled environments in terms
of concentration of particles per volume of air; expressed as the
332 Cleanroom Contamination Prevention and Control

number of particles per cubic meter of air for the size of particles
of interest. To determine the class, a specified testing method is
required, which includes the selection of sampling locations. A major
focus in the revision of the original first edition ISO 14644-1:1999
was the development for a refined statistical approach regarding
the selection and the number of sampling locations. The underlying
assumption in the 1999 version held that particle counts follow a
similar distribution across the room. The new revision discards
the previous assumption in order to allow more accurate sampling
where particle counts may vary in a more complex pattern.

The latest revision of ISO 14644-1:2015 complicates the classific­

ation of ISO Class 5 cleanrooms by the removal of the limit for ≥ 5.0
μm particles. This particle size is referred to as macroparticles and
addressed separately in the informative Annex C. The use of ≥5.0 μm
particles for classification of ISO Class 5 can be accomplished only
by attaching the macroparticle descriptor defining the concentration
limits, specific particle size of interest and the measurement method;
typically, Light Scattering Airborne Particle Counter (LASPC).
Additionally, ≥ 5.0 μm particles can only be used for classification
of ISO Class 5 Cleanrooms when an additional particle size such as
0.5μm is tested. For example:

• Grade B at rest can be expressed as “ISO 5 (29; ≥ 5.0 μm); LSAPC”.

• Grade A at rest and in operation can be expressed as “ISO 5 (20;
≥ 5.0 μm); LSAPC”.

Because particles ≥5.0 μm are more representative of microbial

contamination, non-viable particle monitoring of this size is a part
of a pragmatic contamination control strategy. Characterization
of human borne microbe carrying particles have determined the
average size is 18 µm. (Whyte and Hejab, 2007). In addition, one
cannot assume an underlying distribution of particles in all clean
zones or assume a known relation between 0.5 µm and 5.0 µm
particles (Eaton, 2020).
 Airborne Particle Monitoring Equipment Selection 333

Additionally, the minimum sample volume for classification

is based upon the concentration limits for the largest considered
particle size. When ≥5.0 μm particles are considered for classification
purposes, it increases the sample volume as well as the sample time.
This increase of sample time and volume is exceptionally import­
ant during operational cleanroom classification testing, because the
larger sample provides a more realistic view of the contamination
level during operation. The operational classification testing pro­
vides important data for the risk management program and the
development of an environmental monitoring program.

The elimination of ≥5.0 μm particles for classification per ISO

14644-1:2015 for ISO Class 5 and the requirement for two particle
sizes for both classification and monitoring (0.5 and 5.0 μm) per
GMP Annex 1 has created a confusion.

Due to this an adaptation of the marcoparticle descriptor is in­

cluded in the standard to accommodate the ≥5.0 μm particle size.
ISO 14644-2 emphasizes the need to consider a monitoring strategy
in addition to the classification test requirements of ISO 14644-1. The
requirements of a monitoring plan are outlined, including additional
guidance on the risk assessment as part of the annex to 14644-2.

ISO 14644-1 includes changes that directly impact the require­

ments for light scattering optical particle counters (LSAPC) used in
the pharmaceutical industry. In Appendix F, Test Instruments, it
states that the instrument specifications for the LSAPC are detailed
in ISO 21501-4:2007. Additionally, in Appendix A, it states that the
particle counter used to determine airborne particles shall have a
valid calibration certificate. Furthermore, the calibration frequency
(one year or less) and method should be based upon current
accepted practice as specified in ISO-21501-4 (ISO 14644-1, 2015).

Finally, for sampling of macroparticles (≥5.0 μm) measurement a

minimum flow rate of 28.3 L/min (1 Cubic Foot Per Minute (CFM)) is
a requirement for the particle counters. ISO 14644-2:2015 now clearly
states the use of long sample tubes like those found in sequential
334 Cleanroom Contamination Prevention and Control

manifold systems is not appropriate for monitoring macro particles

(≥5.0 μm).

The 2015 changes to ISO 14644-1 do not necessarily have an

impact on the principles of classification; however, the basis for
classification has been changed from “classification by formula”
(with an informative table to illustrate cleanroom classes) to
classification by a normative table (with a formula for interpolating
intermediate sizes).

Figure 1 is an example table calculated by the formula in ISO

14644-1:1999. The particular sizes and class numbers that are of
most interest in the pharmaceutical industry have been highlighted.
The table also encircles the particle concentrations that have been
removed from the 2015 version of the standard in bolded borders.

Figure 1 ISO 14644:1999 selected airborne particulate

cleanliness classes for cleanrooms and clean zones

Figure 2 is the new classification table from ISO 14644-1:2015, based

on the updated ISO class classification table in ISO 14644-1:2015. This
means the highlighted Annex 1 “maximum permitted particles” in
Figure 3 now fall outside the ISO classification system and therefore
should not be used for formal classification without also including
≥0.5 μm and the macroparticle descriptor for the ≥5.0 μm particle
 Airborne Particle Monitoring Equipment Selection 335

data for Grade A and Grade B cleanrooms. This does not mean that
they cannot be used in an appropriate manner for measurement and
reporting during a classification exercise or for measurement and
assessment during real-time monitoring.

Figure 2 ISO 14644:2015 ISO classes of air cleanliness

by particle concentration

Figure 3 PIC/S Guide to Good Manufacturing Practice for Medicinal

Products. Annexes PE009-12 (Annexes) 1 October 2015
336 Cleanroom Contamination Prevention and Control

Macroparticle descriptor
The ISO 14644-1:2015 revision now describes any particle with an
equivalent diameter ≥5.0 μm as a macroparticle. Where a regulation
requires measurement of these particles, the counting and sizing
of these macroparticles is expressed using the M descriptor in the
format:
ISO M (a; b); c
Where:
a is the maximum permitted concentration of macro particles
(expressed in particles/m³)
b is the equivalent diameter of the macroparticle
c is the specified measurement method (typically LSAPC)

For example, the Grade A at rest concentration of 20 particles/m³ at

≥5.0 μm would be expressed as:
ISO M (20; ≥5.0 μm); LSAPC
The new M descriptor will be used to define the EU GMP Annex 1
Grade A and B and by using the existing ISO 14644-1 designation
of airborne particle concentration (expressed as ISO Class number;
occupancy state; and considered particle size(s)).

Grade A
• ISO 5; at rest, operational; ≥0.5 μm.
• ISO M (20; ≥5.0 μm); at rest, operational: LSAPC.

Grade B
• ISO 5; at rest; ≥0.5 μm and ISO M (29; ≥5.0 μm); at rest: LSAPC.
• ISO 7; operational; ≥0.5 μm, ≥5.0 μm (Grant, 2016).

EU GMP Annex 1 refers to ISO 14644-1 for the purpose of room

classification including the number of sample locations and the
 Airborne Particle Monitoring Equipment Selection 337

required sample size. The ISO 14644-1:2015 version has updates

to classification and sampling, resulting in an important change to
classification. These changes will require companies to redefine their
classification sampling plans and data analysis.

Sampling and classification have been simplified in ISO 14644-

1:2015. A brief summary of changes includes:

• The 95% upper confidence limit (UCL) evaluation for 2–9

sampling locations has been removed.

• Each sampling location is to be evaluated separately and all

sampling locations must pass for a cleanroom or zone to meet
its specification.

• The square root calculation used in ISO 14644-1:1999 to determine

the minimum number of sampling locations has been replaced
with a table. The table provides at least 95% confidence that at
least 90% of the cleanroom or zone will comply with ISO Class
limit. This will in most cases result in an increase in the sample
points compared to the old square root calculation. Note: for
large cleanrooms, e.g., greater than 1000m2 a calculation is still
used.

• The location of the sampling points will now be evenly dis­

tributed at representative locations while avoiding placement
directly under a non-diffused supply air sources in non-
unidirectional cleanrooms.

It is worth mentioning that the statistical accuracy of the sampling

method is based on some underlying assumptions that namely each
location is statistically independent of adjacent locations and that
within each region or location the particle density is homogeneous.
Furthermore, the statistical accuracy assumes that hypergeometric
sampling is used during room certification (Hartvig et al., 2011).
However, the 2015 version of ISO 14644-1 does not require
hypergeometric sampling. Therefore, the standard recommends
338 Cleanroom Contamination Prevention and Control

that additional locations be added if they are known to be critical.

In addition, to mitigate risk, sampling locations may be placed at
the sites of human activity during operation. Air flow visualization
should be used to evaluate the underlying statistical assumptions
of the sampling plan to ensure the desired statistical accuracy is
achieved.

GMP Annex 1 provides additional requirements for non-

viable particle monitoring for both classification and monitoring
(EU GMP, 2008). For classification of Grade A zones, a minimum
sample volume of 1 m3 should be taken per sample location. For
classification purposes portable particle counters with short tube
lengths should be used to minimize the loss of ≥5.0 μm particles
with isokinetic probes. The frequency of reclassification should be
six months for Grade A and Grade B zones and 12 months for Grade
C zones, D zones and all other controlled environments.

Routine monitoring of cleanrooms and clean air devices in

operation is required. Monitoring locations should be based upon
a formal risk analysis including dynamic smoke studies and oper­
ational cleanroom classification test results. For Grade A zones,
particle monitoring should be performed for the full duration
of critical processing including equipment assembly and setup
stages. Grade A zones should be monitored continuously so that
all interventions, transient events, and any system deterioration
can be captured, and alarms triggered if alert limits are exceeded.
For Grade B zones a similar system is recommended although the
sampling frequency may be decreased, however this is not a good
practice because of correlating particle generating events that occur
between Grade A and Grade B environments will be missed if the
Grade B sample frequency does not match that of Grade A. The sig­
nificance of monitoring Grade B zone increases based upon the level
of separation between Grade A and Grade B zones. Considering
human borne contamination should be a prime risk factor during
development of the monitoring parameters in Grade A and Grade
B zones.
 Airborne Particle Monitoring Equipment Selection 339

Airborne particle monitoring systems must be designed to

minimize particle loss when measuring ≥5.0 μm particles by minim­
izing tubing length and the number of tubing bends while making
sure the bend radii is sufficient. ISO 14644-1:2015 states sampling
tubing for ≥5.0 μm particles should be 1 meter or less and the bend
radius of any bend should not be over 15cm.

Figure 4 Examples of a proper and improper bend radius

Source: Microrite, Inc.

Finally, it is worth mentioning that FDA’s Aseptic Guidance for

Industry provides additional requirements for monitoring critical
locations (FDA, 2004). The sampling location in Grade A and ISO 5
zones should be not more than one foot away from the work site
where product is exposed (Figure 5). FDA’s Aseptic Guidance for
Industry recommends the use of remote particle counters over
portable particle counters for continuous monitoring since they are
generally less invasive.
340 Cleanroom Contamination Prevention and Control
Figure 5 The FDA recommends the sample location
be less than 1 foot from the critical area

Source: Microrite, Inc.

AIRBORNE PARTICLE COUNTER TECHNOLOGY

AND SPECIFICATIONS
Light scattering airborne particle
counter technology principle
Light scattering optical particle counters include an optical system
like the one depicted in Figure 6.

Figure 6 Isometric view of a light scattering

airborne particle counter optical system

Source: Microrite, Inc.

 Airborne Particle Monitoring Equipment Selection 341

In Figure 6, the sample air travels through the sensor from the inlet
nozzle to the outlet nozzle as it is drawn through the optical sensor
by a vacuum pump. The optical system includes a light source,
typically a laser diode in a particle counter used in the pharma­
ceutical industry. The laser diode light output is projected into the
sensing chamber by a collimating lens and the main laser beam
is attenuated by a light trap. A particle in the air stream traveling
through the laser beam scatters light in different directions. This
scattered light is collected by the collection optics as in this example
and imaged onto a photodiode. The result is an increase in current
on the photodiode for a few microseconds (the time the particle
is in the laser beam). This current is converted to a voltage pulse
which is processed by counting electronics. The counting electronics
determines the amplitude of the voltage pulse which is related to
the size of the particle through the calibration process defined in
ISO-21501-4. The counter accumulates the number of pulses for each
size channel for a preset sample time. At the end of the sample time
the particles for each size channel are reported as a concentration
(particles/volume of air) based on the flow rate of the instrument
and the sample time.

Light scattering airborne

particle counter specifications
ISO 21501-4:2007 provides a calibration and verification procedure
as well as performance specifications for light scattering airborne
particle counter for clean spaces (ISO 21501-4 2007). ISO 14644‑1:2015
refers to ISO 21501-4:2007 for both instrument specifications and
calibration method and therefore ISO-21501-4:2007 applies to
those particle counters used for non-viable particle monitoring and
classification in the pharmaceutical industry. The following is a
list of parameters that are specified for the particle counter and the
corresponding requirements.
342 Cleanroom Contamination Prevention and Control

• Size calibration
Shall be conducted using calibration particles and an internal or
external Pulse Height Analyzer (PHA) to determine the median
voltage for each calibration size.

An aerosol generator generates calibration particles for each

size channel and the voltage pulses are processed by the PHA
to determine the median voltage of the peak height. The voltage
threshold for each size channel is thus determined.

• Verification of size setting

The size error shall be equal to or less than + 10%.

If the instrument size calibration is rerun, the resulting calibra-

tion size error shall be less than the stated value.

• Counting efficiency
The counting efficiency specification shall be (50 + 20)% for calibration
particles with a size close to the minimum detectable size and (100 +
10)% for calibration particles 1.5 to 2 times larger than the minimum
detectable size.

The count accuracy shall be tested at two particle sizes by

comparing the count values of the instrument under testing to a
count standard which has sufficient sensitivity to measure 100%
of the particles at the sensitivity limit of the instrument under
testing.

This test requires that the calibration setup includes a certified

traceable count standard instrument that is typically one size
more sensitive than the particle counter that is being calibrated.

• Size resolution
The resolution shall be <15% at a size recommended by the
manufacturer.

This parameter is a measure of the optical system’s ability to

resolve two particles that are close to the same size. The sensor
 Airborne Particle Monitoring Equipment Selection 343

resolution is a good indicator of the condition of the optical

system (alignment, cleanliness, light source condition, etc.)

• False count rate

The false count rate is determined by measuring the particle number
concentration in the unit of counts per cubic meter at the minimum
reported size range when sampling clean air.

Typically, this is performed by putting a zero-count filter at the

sample probe location or on the inlet of the instrument.

The false count rate of the instrument quantifies the background

counts recorded due to cosmic radiation, high noise due to
sensor misalignment, contamination build up, etc. Typically,
cosmic ray false counts only affect the smaller size channels and
should not be a factor in pharmaceutical industry applications.
On the other hand, sample tube and sensor cleanliness can be a
factor as large particles flake off surfaces periodically.

Since the time of arrival of the false counts is random, histori-

cally the zero-count specification for a particle counter has been
described assuming Poisson statistics and specified at the 95%
upper control limit (UCL) of the distribution. Therefore, if a par-
ticle counter has a zero-count rate of one particle every five min-
utes, statistically the instrument could fail one out of every 20
(5%) zero count tests and still be in compliance. Still, this test is a
very important confirmation of performance that is required per
ISO-14644-1:2015 testing protocols.

• Maximum particle number concentration

The maximum measurable particle number concentration shall be
specified by the manufacturer. The coincidence loss at the maximum
particle number concentration of an LSAPC shall be equal to or less
than 10%.

Optical particle counters are single particle counters and are

used at concentrations where there is a very small probability
of two particles passing through the optical sensor at the same
344 Cleanroom Contamination Prevention and Control

time. The maximum particle number concentration specifies the

upper concentration limit at which the probability of coincidence
count errors is no longer negligible (e.g., 10% error equivalent to
10% of a cleanroom class).

In optical particle counter design, there are two types of

coincidences. The first is optical coincidence and is a function of
the sensing volume. The second type of coincidence is electronic
coincidence determined by the speed of the pulse processing
electronics. In practice, detecting coincidence is extremely
difficult in the field. Therefore, it is good practice to make sure
the instrument is operating well below its concentration limit to
maximize its count accuracy.

• Sampling flow rate

The standard uncertainty of volumetric flow rate shall be equal to or
less than +/– 5%.

The flow rate in a portable particle counter such as those used

for cleanroom certification is typically controlled by varying the
pump performance based on sensor feedback in real time. The
flow rate of a remote particle counter used in a real time Grade A
monitoring system may be set by a critical orifice at the output
of the sensor if a central vacuum system is used for air sampling.

• Sampling time
The standard uncertainty of the sample time shall be less than +/- 1 %
of the preset value.

This parameter should be validated as part of any product

design and will ultimately affect the particle concentration
proportionally. Typically sampling time inaccuracies are insig­
nificant in a proper design.

• Response rate
The response rate of the LSAPC obtained according to the test method
given shall be less than 0.5%.
 Airborne Particle Monitoring Equipment Selection 345

This parameter is tested by first measuring particles near the

concentration limit then changing the measurement to clean air
(0 particles). The ratio of counts should be less than 0.5% from
at least a particle level of 1000 counts. The sample time should
be 60 seconds or less and a 10 second interval is used for the
change from high concentration to zero particle air samples.
The sample tube, inlet geometry and optical cavity can all play a
role in the response rate.

• Calibration interval (frequency)

It is recommended that the calibration interval of the LSAPC be one
year or less based upon current practice as specified in ISO 21501-4.

• Test report
The following minimum information shall be recorded.
– Date of calibration.
– Calibration particle sizes.
– Flow rate.
– Size resolution (with particle size used).
– Counting efficiency.
– False count rate.
– Voltage limit or channel of an internal pulse height analyzer.

The ISO 21501-4 requirements provide a fairly comprehensive

list of performance requirements for an instrument to be
validated during the engineering design project for the product.
In addition, the calibration interval and test report provide a
very clear set of requirements for the periodic recalibration of
the instrument to assure its accuracy over time.
346 Cleanroom Contamination Prevention and Control

OPTICAL PARTICLE COUNTERS

TYPES AND APPLICATIONS
Handheld particle counters
Handheld particle counters like the one in Figure 7, have a flow rate
of 0.1 CFM and are therefore not acceptable for use when measuring
large particles (e.g., 5.0 µm). They have the advantage of being light­
weight and small and additionally have a higher concentration limit
than higher flow rate particle counters. They can be considered for
some applications like leak detection and recovery testing where
they may be more sensitive due to their ability to count higher
concentrations and not saturate when measuring extremely high
particle concentrations.

Figure 7 Handheld particle counter

Source: Courtesy of TSI Inc.

Portable particle counters

Classification of cleanrooms can be conducted with portable particle
counters or portable particle counters in combination with the
dedicated remote particle counters that are used for continuous
monitoring. Classification should be performed with short sample
tubes to maximize the accuracy of the ≥5.0 μm particle measurement.
Ideally a fixed isokinetic sample probe will mount directly to the
 Airborne Particle Monitoring Equipment Selection 347

input of the optical particle counter when classifying unidirectional

cleanrooms with HEPA filters mounted in the ceiling and the airflow
is in the vertical direction. Figure 8 is a picture of a portable particle
counter connected to an isokinetic probe mounted on a probe stand
via 1 meter of tubing. The advantage of using a probe stand is that
one can easily orient the probe in the direction of horizontal flow in
the case of a horizontal laminar air flow hood.

Figure 8 Portable particle counter

Source: Microrite, Inc.

Because ISO 14644-1:2015 also stipulates a minimum flow rate of

1 CFM when measuring ≥5.0 μm particles and EU GMP Annex 1
requires a sample volume of 1m3 for Grade A zones there has been
the advent of higher flow rate particle counters to minimize sample
times in these applications. For example, a 100 liter per minute
(LPM)flow rate will reduce the sample time from approximately
35 minutes with a classic 1 CFM to only 10 minutes. However,
consideration should be given to other factors including the
concentration limit of the instrument. For Grade C, in operation,
and Grade D, at rest, measurements of the concentration limit for
the zone equates to approximately 100,000/ft3. This concentration is
very close to the concentration limit of a 100 LPM particle counter
where the additional error is already 10%. Furthermore, this limit
is not tested at calibration periodically and the limit is difficult for
348 Cleanroom Contamination Prevention and Control

the manufacturer to completely validate. Finally, concentration

limit errors are not easily identified in the data and are generally
not detectable by the measuring instrument. Hence, there is a
greater risk of measurement errors occurring when using a particle
counter to measure concentration levels that are approaching the
instrument’s concentration limit. For this reason, the concentration
is typically set to one fifth of the concentration limit of the instrument
for critical tests like size calibration and count efficiency accuracy
checks by the manufacturer. Ideally, the target class limit being
measured would also be on the order of one fifth of the instrument’s
concentration limit to maximize the accuracy of the Class or Grade
limit measurement. In summary, where maximum accuracy and
minimum risk is required, the ideal flow rate of a particle counter
used for classification of all Grade zones would be 1 CFM. This will
maximize the accuracy of measuring ≥5.0 μm particles in all clean
zones in pharmaceutical manufacturing.

Another advantage of using a 1 CFM particle counter in

Grade A locations is that the sample time is maximized to assure
the measurement is the most accurate representation of particle
levels under dynamic operating conditions. Finally, using a 1 CFM
flow rate particle counter for classification has the added advantage
of matching the portable particle counter’s flow rate to the flow
rate of the remote particle counters utilized for online continuous
monitoring of aseptic processing. This practice will result in the
best correlation between classification test data and monitoring data
under dynamic operating conditions when localized variations in
particle concentrations are likely and particle concentrations matter
the most to product quality. In fact, periodic reclassification testing
can incorporate data from the continuous monitoring system to best
classify the Grade A zone during dynamic operating conditions. At
the same time this practice may offset the extra time required for
reclassification with a 1 CFM particle counter instead of a 100 LPM
instrument, since these continuous monitoring locations are always
being measured simultaneously.
 Airborne Particle Monitoring Equipment Selection 349

Remote particle counters

Fixed location remote particle counters are recommended where
continuous monitoring is required. This includes Grade A zones as
defined in EU GMP Annex 1 and Critical Areas as defined in the
FDA’s Aseptic Guidelines. The regulations require the instruments
have a flow rate of 1 CFM. Fixed location remote particle counters
minimize the impact of the continuous monitoring system on the
process and environment. Small remote particle counters can be
located close to the process, thereby facilitating the use of short,
straight sample tubes between the probe and sensor to accurately
monitor ≥5.0 μm particles. Figure 9 is an example of a remote particle
counter.

Figure 9 Remote particle counter

Source: Courtesy of TSI Inc.

Historically remote particle counters make use of a centralized

vacuum pump located remotely for air flow. A critical orifice at
the outlet of sensor creates choked flow at the desired flow rate as
long as sufficient vacuum is present. This minimizes the footprint of
the sensor, frequently allowing it to be located closer to the sample
location. In these applications the sensor is typically housed in a
stainless-steel enclosure to facilitate wipe down. If the location is
subject to VHP decontamination the sensor flow path and exterior
should be designed to be impervious to VHP.
350 Cleanroom Contamination Prevention and Control

Remote particle counters with integrated pumps have also been

used in these fixed-point installations for monitoring. However, they
have a larger footprint than conventional remote particle counters
which can make it difficult to optimize the sample tube length and
geometry. Additionally, consideration needs to be given to the
location of the pump exhaust of these instruments, so the exhaust
air flow does not affect the unidirectional clean air flow in critical
areas. Figure 10 is a picture of a remote particle counter with a built-
in pump.

Figure 10 Remote particle counter including an integrated pump

Source: Courtesy of TSI Inc.

The reliability of remote particle counters is critical in continuous

monitoring applications. For this reason the most advanced instru­
ments and systems now incorporate extensive diagnostics for laser
output levels, sensor cleanliness, flow rate, calibration expiration,
communication status, etc. to assure that each particle counter is
accurately monitoring the operation at all times.

Continuous monitoring systems include facility monitoring

software that often incorporates input from other sensors includ­
ing temperature, relative humidity, differential pressure and
air velocity measurement devices. The software should be fully
compliant with the FDA 21 CFR Part 11 standard which applies to
records in electronic format that are created, modified, maintained,
 Airborne Particle Monitoring Equipment Selection 351

archived, retrieved, or transmitted according to requirements set

in FDA regulations. Electronic records/signatures that meet Part 11
requirements may be used in lieu of paper records.

Common mistakes when selecting, installing

and operating optical particle counters
The following pitfalls should be avoided when selecting and install-
ing optical particle counters.

• Specifying the incorrect flow rate instrument (e.g., a 0.1 CFM

handheld particle counter for cleanroom classification or envi-
ronmental monitoring).

• Selecting a particle counter that emits particles into the clean­

room (possibly through the air flow exhaust or cooling fan
exhaust).

• Permitting the exhaust of the vacuum source (pump, etc.) to

affect the air flow in an critical location.

• Using a sample tube that is too long or contains too many bends.

• Selecting the wrong size channels.

• Not specifying ISO 21501-4 calibration.

The following is a list of common mistakes made when operating

optical particle counters.

• Spraying IPA (70% Isopropyl Alcohol) in the vicinity of the

isokinetic probe resulting in higher than actual counts.

• Performing other activities close to the monitoring point leading

to higher counts due to personnel shedding.

• Inappropriate sanitization and wipe down of particle counters.

352 Cleanroom Contamination Prevention and Control

• Inadequate “zero” count due to contaminated sample tubes or

other issues.

• Incorrect sample volumes or sample times for continuous

monitoring.

• Incorrect sample volumes or sample times for certification

testing.

• Incorrectly normalizing of counts to the appropriate concen­

trations as specified in the standards and regulations.

In summary, optical particle counters are sensitive optical systems

that must repeatably, reproducibly and accurately measure the
process and environment to mitigate risk of product contamination.
Therefore, it is critical that these instruments are specified, installed
and operated correctly.

INDUSTRY STANDARDS, GUIDELINES

AND PRACTICES FOR VIABLE
PARTICLE MONITORING
The main sources for guidance related to viable particle monitoring
and setting limits include EU/ GMP Annex 1, and FDA 2004 Aseptic
Guideline. The current draft version of EU/ GMP Annex 1 has a
specific section on viable and non-viable monitoring and proposes a
risk-based approach including use of smoke studies for selecting of
monitoring sites. Figure 11 shows the viable particle limits for each
zone/class for both the EU GMP Annex 1 and FDA guidelines.

USP non mandatory Chapter <1116> “Microbiological control

and monitoring of aseptic processing environments” provides
additional guidance and more details related to an environmental
monitoring program (Sandle, 2013).
 Airborne Particle Monitoring Equipment Selection 353
Figure 11 Microbial limits during operation, according
to European Union Guidelines (Annex 1) (top) and
FDA Guidance (2004) (bottom)

There are multiple sampling techniques that can be used to monitor

viable particles in aseptic processing. At present, nearly all of these
methods rely on the growth and recovery of microorganisms many
of which may be in a damaged state due to the environment and
therefore may not be recoverable. After collection, the samples are
incubated and the resulting colony forming units (CFUs) are counted.

USP Chapter <1116> regards all types of viable environmental

monitoring as semi-quantitative given the following:

• The methods are inaccurate.

• Variation exists between methods (for example, models of active

air samplers vary in collection efficiency up to a factor of 10).

• Recovery of microorganisms can be low, with swabs and contact

plates recovering less than 50% of the microbial population
found on a surface.
354 Cleanroom Contamination Prevention and Control

• People can contaminate any sample through the act of taking

the sample.

• The methods are poor at recovering damaged or stressed micro-

organisms (like those found in aseptic filling environments).

Furthermore, several studies have demonstrated that there are a

large proportion of microorganisms that are viable but do not grow
on standard agar media (viable but not culturable). Hence these
microorganisms are not detected using conventional techniques.

In addition, the traditional sampling methods do not sample

everywhere and all the time. Therefore, these measurements are not
a way to guarantee the sterility of product but rather a method to
indicate the manufacturing process is in control. Though automation
in viable particle monitoring is used as an exploratory tool, growth of
viable organisms remains a preferred method to assess the number
of CFUs and also the type of organisms recovered.

When alert and action limits are exceeded, the typical microflora
should be identified in order to determine the risk posed by the
detected contamination identified and help identify the root cause.
Organisms such as Staphylococcus aureus can enter the cleanroom
via people’s skin (O’Donoghue, 2017). Bacillus species are spore
forming and difficult to remove and can enter the cleanroom via
foot or wheel borne traffic, as well as on materials/items transferred
into the room which are not cleaned effectively. Gram-negative
bacteria must never enter the cleanroom as they produce endotoxins
that cannot be removed by sterilization techniques. Such organisms
could compromise patient health even leading to a fatality. Water is
the most common source of these types of organisms. As can be seen
from these examples, the identification of the type of organism can
aid greatly in determining root cause and corrective action before
product contamination occurs.

Hence efficient recovery of viable particles is critical to assessing

risk of environmental isolates to the quality of product.
 Airborne Particle Monitoring Equipment Selection 355

Viable air sampler technology

A volume of air for viable sampling is collected and impacted onto
agar medium. The medium with the impacted microorganisms
is incubated at appropriate temperatures to recover the impacted
organisms. Viable microorganisms form CFUs as they multiply.
These CFUs are reported, and as appropriate also characterized.

Centrifugal impaction
Centrifugal samplers draw air into the front of the sampler by a
rotating vane. Centrifugal force then throws the microbe-carrying
particles onto a nutrient agar surface. The agar surface is in the form
of a strip.

Slit-to-agar
Slit-to-agar (STA) samplers sample through a slit onto a slowly
rotating Petri dish with the nutrient agar. The operator is thus able to
determine when the contamination occurred based on the location
of the CFU on the agar. The flow rate is 1 CFM and the exhaust is
HEPA filtered.

Sieve impaction
Sieve impaction draws air through perforated plates. Micro­
organisms are impacted onto an agar surface held in a Petri dish.
Air flows vary between 25, 50 and 100 LPM, and for portable type
instruments the exhaust is HEPA filtered. Portable devices are
appropriate for monitoring conventional cleanroom environments.
There are also remote devices for monitoring Grade A and critical
locations including isolators and RABS. These devices are stainless
steel and can be sanitized and the stainless sample heads can be
sterilized. They contain an outlet port and typically a critical orifice
to connect to a remote vacuum pump and provide the specified flow
rate through the device. Figure 12 shows an example of a remote
356 Cleanroom Contamination Prevention and Control

active air sampler with integrated flow measurement to validate the

sample volume in real time.

Figure 12 Remote active air sampler

Source: Courtesy of TSI Inc.

Other sampling devices are filtration devices which sample air

onto membrane or gelatin filters that are then contacted to the agar
prior to incubation. Impingers use a liquid medium for particle
collection. Low shear-force liquid impingers can recover stressed
organisms and thus claim the best recovery rates for many airborne
microorganisms. However, these devices are not very portable,
have long measurement times and require additional laboratory
expertise to process. Cascade sieve samplers can provide particle
size distribution while STA samplers provide time resolution of
viable particle measurement. Settling plates are useful to locate point
sources of large particle contamination. Centrifugal and several
types of sieve samplers provide a simple, rapid method of active air
sampling where viable particle size and temporal information are
not required.

Two main properties of an active air sampler effect the

instrument’s ability to capture and assist in incubation of the
captured particles on a media plate (Kelley, 2017).
 Airborne Particle Monitoring Equipment Selection 357

Physical efficiency – the ability of the air sampler to collect various

sizes of particles. This efficiency is the same whether the particle is a
microorganism, carries a microorganism or is an inanimate particle.
Physical efficiency is based on many factors and the sample head
geometry and air sampler internal design, including the media height,
even environmental conditions will influence physical efficiency.

Biological efficiency is the efficiency in collecting microbe-carrying

particles. Biological efficiency will be lower than physical efficiency
for a number of reasons, such as the survival of the microorganisms
during collection and the ability of the collection medium to support
their growth.

Validation of impaction type active air samplers is carried out

in accordance with ISO-14698 Appendix B. The method suggests
the physical efficiency should be measured using Bacillus atrophaeus.
These spores are robust and unaffected during sampling by drying or
stress; they can therefore be used to measure the physical efficiency of
air samples. Other test particles including polystyrene latex spheres
or dye particles can be used. Measuring the biological collection
efficiency is less reliable because drying and stress problems make
it difficult to be sure of the actual concentration in the test chamber.
If the biological efficiency is measured, a microbe should be used
that is typical of those found in cleanrooms, i.e., Staphylococcus
epidermidis. Particles are generated in a chamber and monitored with
a membrane filter. The counts from the sampler being tested and
the membrane filter are compared over five sizes spread between
0.6 µm and 15 µm to calculate the physical efficiency at each size. It is
important to understand that the test method requires an apparatus
unavailable in a routine microbiology laboratory and should be
carried out by an independent test laboratory. The validation test
results should be provided by the manufacturer of the air sampler.

In addition, periodic calibration of the air active sampler sample

volume is required. Lastly, testing should be conducted to assure
desiccation of the agar medium does not occur during the length of
the sample and subsequent incubation period.
358 Cleanroom Contamination Prevention and Control

According to ISO 14698, there are many factors to consider when

choosing an air sampler. The sampling rate, duration of sample
and type of sampling device can strongly influence the viability of
the microorganisms that are collected. Because of the number and
variety of microbial air sampling systems commercially available,
the selection for a particular application should consider, as a
minimum, the following factors:

• Type and size of viable particles to be sampled.

• Sensitivity of the viable particles to the sampling procedure.
• Expected concentration of viable particles.
• Ability to detect high or low levels of bio-contamination.
• Appropriate culture media.
• Time and duration of sampling.
• Ambient conditions in the environment being sampled.
• Disturbance of unidirectional airflow by sampling apparatus.

One of the most fundamental selection criteria is the resolution of

the air sampler which can be equated to what is known as the d50
cut-off point. This value can be calculated by design parameters
and is validated by measuring the physical collection efficiency as
described above at multiple particle sizes above and below the d50
cutoff point.

It is important to select an air sampler with the right d50

(resolution) and ability to collect the smallest particle size feasible,
to ensure confidence in the results.

D50 is based on particles greater than a certain aerodynamic size

collected on the media plate and particles less than that size passing
through the air sampler.

The sample head must be capable of effectively capturing

particles in the air and maintaining unidirectional conditions
 Airborne Particle Monitoring Equipment Selection 359

between the sample head and the media where the particles impact.
Smaller particles are subject to airflow and larger particles maintain
their flight path due to higher inertia. The d50 is the point where
50% of the smallest size particles impact on the media, in other
words, it is really the resolution of the impactor as the other 50% of
these smaller particles will follow the airflow and not impact on the
media.

The same holds true for remote sampling heads used in auto­
mated systems for continuous monitoring where a remote vacuum
source is controlled using mass flow meters for volume control.

The following method has been suggested on how to design

impactors (Whyte et al., 2007).

• Ensure that the Reynolds number of the air passing through the
nozzle is between 500 and 3000.

• Select and appropriate d50 “cut-off” of particle, calculate and

select the variables required to achieve this cut off sizes. This
can be done either via using Stokes number or the “simplified
analytical method” employed by others.

• Ensure that the ratio of the separation distance (S), sometimes

referred to as the nozzle to surface distance, to the diameter
or width of the nozzle (W) is not less than 1.5 in rectangular
nozzles and 1 in round nozzles. This rationale is given by a
dimensionless number that is generally known as the S/W ratio.

• If possible, the entrance to the nozzle should be tapered or

conical, and of constant width or diameter, with a nozzle throat
length at least as large as the width or diameter of the nozzle.

CFD analysis has been performed on a validated sampler. CFD results

showed a slightly larger cut off than the simplified analytical model
and this result emphasizes the need to validate the physical efficiency.
360 Cleanroom Contamination Prevention and Control

At the same time the biological efficiency needs to be understood

because some parameters that will improve the physical efficiency
(e.g., velocity) may have an adverse effect on the biological efficiency
if they cause damage to microorganisms.

The overall efficiency of the sampler is a function of the d50 cut

off and the distribution of viable particles in cleanrooms.

Since the majority of viable particles in cleanrooms are generated

by humans and controlled by cleanroom garments, substantial
research and modeling has been conducted on the particle size
distribution of these particles. A compilation of these results shows
that the size distribution of microbe-carrying particles in occupied
rooms conforms well to a log-normal distribution, with an average
equivalent diameter of 12 µm, 25% and 75% being large than 20 µm
and 4 µm respectively. In addition, microbes sampled in cleanrooms
are mainly bacteria with a small proportion of spores, these micro-
organisms have a unicellular size that is unlikely to be much less
than 1 µm, and at that size a frequency of occurrence of less than 1%.
Additionally, testing of particle and microbial airborne dispersion
from people using an Andersen sampler has been used to confirm
the average particle sizes of those dispersed by humans. Thus, when
reviewing the overall efficiency of air samplers, it is apparent that if
the d50 it is much greater than 1 µm it will have a significant effect
on the efficiency of measurement of micro-carrying particles in
occupied cleanrooms.

The overall efficiency of multiple samplers with similar and

different d50 cutoffs have been documented by controlled testing of
particle generation from humans wearing cleanroom garments. The
tests demonstrate that air samplers with similar d50 values below
2 µm track changes in particle levels dispersed from operators due
to the quality of cleanroom garments with similar CFU/m3 values
reported by both instruments (Romano et al., 2014).

Significantly different results (5–10 times difference) were

reported when testing the microbiological contamination generated
 Airborne Particle Monitoring Equipment Selection 361

by two people with two different air samplers with different d50
cut off values (e.g., <1.65 µm versus <14.44 µm). Furthermore,
testing of multiple active air samplers with different d50 cutoffs
has demonstrated significant differences when measuring microbe-
carrying particles at statistically significant concentrations. (Whyte,
2005).

Additionally, the flow rate accuracy can affect the volumetric

accuracy of the instrument. Some manufacturers specify the flow rate
accuracy at +/- 2/5% others specify +/- 10%. When sampling 1 cubic
meter this can result in a 75 liter difference in volume sampled by
different instruments (Sandle, 2010).

Finally, below are some key attributes of best-in-class active air

samplers.

Air sampler attributes

• Physical size – small footprint.
• Construction material for enclosure/sample head – preferably
stainless steel.
• Ability to wipe down easily – no crevices, buttons switches or
particle traps.
• Media plate holder – easily adjustable holder mechanism, media
dish diameters vary +/- 1 mm to 3 mm.
• HEPA filtered exhaust – captures viable particles that have not
impacted.
• Touchscreen interface – reduces contact and potential particle
generation.
• Battery operated for better portability on portable units.
• Remote sample options – offer more flexibility.
• Gas connector options for testing gases to ISO 8573 requirements.
362 Cleanroom Contamination Prevention and Control

• Local or field calibration options from supplier.

• Easily autoclaved parts.
• Particle physical collection efficiency (d _50) < 2 micron (µm) per
ISO 14698 and EN 17141.
• Validated for collection efficiency by third party.

Figure 13 shows an example of a portable active air sampler suitable

for use in aseptic processing.

Figure 13 Portable active air sampler

Source: Courtesy of Lighthouse Worldwide Solutions, Inc.

CONCLUSION
Selection of viable and non-viable monitoring equipment is key to
obtaining reliable data during environmental monitoring. Environ­
mental monitoring data is representative of the facility’s condition,
gowning practices, aseptic techniques and efficacy of contamination
control measures such as air quality, airflows, material flows,
personnel flows, waste flows as well as disinfection and cleaning.
Due diligence when selecting appropriate equipment is paramount
to assessing product and patient risk.
 Airborne Particle Monitoring Equipment Selection 363

REFERENCES
21 CFR. Part 11-Electronic records; electronic signatures.

BS EN 17141:2020 Cleanrooms and associated controlled environ-

ments. Biocontamination control.

Eaton, T. (2020) Pharmaceutical Cleanroom Classification Using ISO

14644-1 and the EU GGMP Annex 1 Part 2: Practical Application.
EJPPS European Journal of Parenteral and Pharmaceutical Sciences
244 (2020): 17–36.

EU GMP Annex 1 2020 draft https://ec.europa.eu/health/sites/health/

files/files/gmp/2020_annex1ps_sterile_medicinal_products_en.pdf

EU GMP (2008) Annex 1 – Manufacture of Sterile Medicinal Products.

FDA (2004) Guidance for Industry. “Sterile Drug Products Produced

by Aseptic Processing – Current Good Manufacturing Practice.”

Grant (2016) What does the recent ISO 14644-1:2015 update mean
for pharmaceutical cleanroom classification. Pharmaout.net 3 18.

Hartvig, N.V., Gordon, J., Farquharson, R.M., Varney, M., Foster, M.

(2011) Sampling plan for cleanroom classification with respect
to airborne particles. Journal of the IEST 54 (1): 1–15.

ISO (2015) ISO 14644-1. Cleanrooms and associated controlled

environments – Part 1: Classification of air cleanliness by particle
concentration.

ISO (2015) ISO 14644-2. Cleanrooms and associated controlled

environments – Part 2: Monitoring to provide evidence of
cleanroom performance related to air cleanliness by particle
concentration.
364 Cleanroom Contamination Prevention and Control

ISO (2007) ISO 21501-4. Determination of particle size distribution -

Single particle light interaction methods – Part 4: Light scattering
airborne particle counter for clean spaces.

ISO (2003) ISO 14698-1. Cleanrooms and associated controlled

environments – Biocontamination control – Part 1: General
principles and methods.

Kelley, J. (2017) Selecting portable and automated air sampler

devices to meet cGMP. Cleanroomtechnology.com March.

Kelly, J. (2005) “Pharmaceutical Industry Cleanroom Monitoring:

Viable and Non-Viable Particle Detection.” rdmag.com January.

O’Donoghue, K. (2017) “Validating and Monitoring the Cleanroom.”

In Handbook for Critical Cleaning: Applications, Processes, and
Controls. pp. 89–102.

Romano, F., Gusten, J., Joppolo, D., Ljungqvist, B., Reinmüller, B.

(2014) Some aspects on the sampling efficiency of microbial
impaction air samplers. Particuology 20: 110–113.

Salaman-Byron, A.L. (2018) Limitations of microbial environmental

monitoring methods in cleanrooms. American Pharmaceutical
Review April.

Sandle, T. (2013) New guidance for environmental monitoring in

cleanrooms: an overview of the revised USP <1116> chapter.

Sandle, T. (2010) Selection of active air samplers. European Journal of

Parenteral and Pharmaceutical Sciences 15: 119–124.

USP (2012) <1116>. Microbiological Control and Monitoring of

Aseptic Processing Environments. May.

Whyte, W., Hejab, M. (2007) Particle and microbial airborne

dispersion from people. European Journal of Parenteral and
Pharmaceutical Sciences 12(2): 39–46.
 Airborne Particle Monitoring Equipment Selection 365

Whyte, W., Green, G., Albisu, A. (2007) Collection efficiency and

design of microbial air samplers. Journal of Aerosol Science 38(1):
97–110.

Whyte, W. (2005) Collection efficiency of microbial methods used

to monitor cleanrooms. European Journal of Parenteral and
Pharmaceutical Sciences 10(2): 3–7.

ABOUT THE AUTHOR

Bill Shade possesses extensive engineering experience in advancing
of particle counting technology through innovative product design
and project management resulting in full product development
lifecycles. His engineering background includes applications,
systems, optical and electrical design. Bill has broad experience
in troubleshooting particle counting/generation devices as well as
configurations including particle monitoring systems and smoke
generating devices. He has developed new technologies in particle
monitoring science; he has also created innovative optical designs of
high flow particle counter sensor using a refractive mirror resulting
in the smallest high flow particle counter sensor design to date.
 12

FOOT AND WHEEL BORNE

CONTAMINATION

Krishna Bell
Acumen Technology, Inc.
Blaine, MN, USA
Ziva Abraham
Microrite, Inc.
San Jose, CA, USA

The control of foot and wheel borne traffic into and out of cleanrooms
and other controlled areas is an important part of contamination
control. It is critical to understand the difference between human
borne contaminants generated by personnel working in the
cleanrooms versus the contamination brought in from the outside;
this differentiation will highlight the significance of preventing
ingress of soil borne contamination from outside the cleanrooms.

Contamination sources of concern from humans working in

cleanrooms relates to organisms generated from exposed body parts
after gowning. Microorganisms generated by personnel working in
cleanrooms include both aerobes as well as anaerobes.

367
368 Cleanroom Contamination Prevention and Control

Organisms prevalent on the skin include S. epidermidis, a major

inhabitant of the skin. Other organisms prevalent on the skin include
staphylococci, micrococci and diphtheroids such as corynebacteria
(Davis, 1996).

Anaerobic organisms are most common in areas rich in

sebaceous glands; organisms such as Propionibacterium acnes and P.
granulosum also constitute skin flora. P. acnes is seen more frequently
than P. granulosum.

Staphylococcus aureus is prevalent in the nose and often present

on the skin of patients with certain dermatologic diseases. On the
other hand, streptococci, such as α-hemolytic streptococci, exist
primarily in the mouth, from where they may, in rare instances,
spread to the skin.

Gram-negative bacteria make up a small proportion of skin

flora. Acinetobacter spp. is among the most commonly found on the
skin of normal individuals (Davis, 1996).

Oral flora constitute primarily those bacterial genera found in

the normal oral cavity (for example, α-and β-hemolytic streptococci);
however, anaerobes, staphylococci, neisseriae, diphtheroids, and
others are also present. Human borne organisms, whether aerobes
or anaerobes are easily killed using sanitizers such as 70% Isopropyl
alcohol (IPA), hydrogen peroxide or general-purpose disinfectants
such as phenolic or quaternary ammonium compounds.

In contrast to the type and level of contamination introduced

by personnel, foot and wheel borne traffic transport a plethora of
organisms that are not as easy to eliminate as human borne flora,
utilizing general purpose disinfectants or sanitizers.

Bacteria and fungi, among other organisms in the soil, actively

participate in organic matter decomposition. Microorganism
numbers and types vary depending upon soil types and soil moisture
content. The counts of bacillus (spore forming Gram-positive rods),
 Foot and Wheel Borne Contamination 369

Gram-negative bacteria, actinomycetes and fungi, with bacteria

being the most numerous, may be different in moist soil, dry soil,
agricultural soil, forest, etc. In general, organism counts range from
4×106 to 2×109 per gram (Vieira and Nahas, 2005).

Some spore forming bacteria and many mold spores are hard
to eliminate even when utilizing sporicidal disinfectants such as
sodium hypochlorite and peracetic acid chemistries (PAA). In recent
years there has been a heightened awareness of mold contamin­
ation by regulators and industry alike. Mold varies from those
such as Penicillium or Aspergillus which can be killed by general
purpose disinfectants such as phenolic and quaternary ammonium
compounds and sometimes sanitizers such as 70% IPA. On the
other hand, many mold species which may enter the cleanroom via
tracked soil, or those that reside on paper, cardboard or pallets are
often difficult to eliminate.

Personnel and material traffic from uncontrolled areas or the

warehouse can be transported on shoes or wheels of material carts.
This is where leaving contamination outside the cleanrooms has
been one of the key elements of contamination control. Addition­
ally, dispersal of particles, including microorganisms, in turbulent
flow clean zones or controlled environments occurs relatively
easily (Sandle, 2012). Eventually any particles present will be
either removed from a clean area, through the function of the air-
handling system, or deposited onto a surface because of gravity,
static, convection, or diffusion. Once contact has been made with
a surface, particles will adhere to the surface either “reversibly”
(i.e., temporarily) or “irreversibly” (i.e., permanently) through a
combination of chemical or electrostatic forces.

The goal of the tacky mat or polymer flooring, whichever the

company chooses, is not just for removal of dirt and debris from
operator shoes or cartwheels, but also to ensure that the removed
dirt stays on the tacky mat or polymer flooring and does not
become airborne.
370 Cleanroom Contamination Prevention and Control

TEMPORARY AND PERMANENT STICKY MATS

A sticky mat, also known as tacky mat or peel-off mat, is a mat with
an adhesive surface that is placed at the entrances or exits to certain
areas to remove contaminants from the bottoms of footwear and
wheeled carts. Sticky mats can be temporary or semi-permanent.
Temporary sticky mats are made of a stack of adhesive plastic
film layers that are periodically peeled off and discarded. Semi-
permanent mats consist of a polymer layers with mesh backing,
containing no adhesives, these are cleaned and dried with a mop or
a squeegee.

Temporary sticky mats

Temporary sticky mats have been in use in the industry for a long
time, they provide a good contamination control effect if used
properly. Though they are designed to be contamination removal
step when entering a cleanroom and a measure for preventing
contamination in the more critical areas such as gown rooms,
if not properly used, can become a contamination source and
contamination transport vehicle.

Common mistakes made when using temporary sticky/tacky

mats that have led to contamination events include the following:

• Placement
Placement of tacky mats is key. The purpose of tacky mats is
to remove contamination from soles of the shoes and wheels
of the carts before entering the controlled environment. Often,
companies become overzealous and place mats inside the
gowning areas, at the entrance to the clean corridors, or even at
the entrances of clean zones. It is important to understand that
using tacky mats after donning shoe covers or dedicated shoes
is not conducive to contamination control (Estes et al., 2019).
The surfaces of these mats are sticky and some use aggressive
adhesives. The soles of single use shoe covers can easily become
compromised by stepping on the adhesive surface of the mat,
 Foot and Wheel Borne Contamination 371

hence compromising their integrity and turning the soles into

sieves through which contamination can easily escape. Even if
dedicated shoes are utilized, they can build up adhesive on the
surface of the shoe dragging more contaminates into the clean
areas.

• Size
On many occasions, the tacky mat is not long enough for a
person’s stride, causing him/her to step on it with only one
foot and not the other. An ideal situation for contamination
removal is to install a sticky mat long enough for two to three
steps with each foot. That way, one can guarantee that soil and
debris has been reduced considerably. The size of the sticky mat
is important for it to serve its purpose. It should be ensured that
the mat is wide enough to accommodate most traffic; the wider
the better.

• Adjustment
If the adhesive mat is the size of a doormat, instead of placing
it horizontal to the door, lengthwise placement, in the path
of traffic, may help in getting personnel to step on the mat
multiple times.

• Peeling
The peeling process for temporary mats, depending upon how
the mats are peeled, may dislodge particles from the mat; these
particles may fall around the mat or may become airborne. The
airborne particles can be carried into the cleanroom (Sandle,
1970).

• Use
Routine change of tacky mats should be ensured. Many environ­
mental monitoring excursions for surface as well as active
monitoring in the gown rooms are due to inappropriate practices
when using and changing tacky mats. This is a contamination
issue which is often overlooked when investigating environ­
mental monitoring excursions.
372 Cleanroom Contamination Prevention and Control

• Audit
It is important to have a mat audit program for the reasons
mentioned above as well as monitoring for mold growth around
the edges where the mat is adhered to the floor. Mold is known
to grow on glue, labels, and other surfaces such as the glue that
is used to adhere the mat to the ground.

• Cleaning
Each time a new stack of mats is installed ensure removal of any
old adhesive on the floor. Adhesive, as mentioned above, is an
attractant for mold which can proliferate over the lifecycle of the
new mat stack; this type of contamination can then inadvertently
be carried into the controlled environment. It is important to
make certain that the floor is completely dry before installing a
new sticky mat as moisture my promote mold growth as well.

• Color
Adhesive mats come in both light and dark colors. The choice
is up to the user, however, lighter colored mats make dirt and
debris move visible to the naked eye. This promotes proactive
mat peeling and replacement.

Semi-permanent sticky mats

Semi-permanent sticky mats, otherwise, also known as polymer
flooring systems, are an alternative to temporary sticky mats and
are a welcome solution to contamination control programs. Human
traffic adds the most contamination to controlled areas. Using
polymer flooring systems to control transitions from less clean
areas to more controlled areas reduces contamination risk. A study
conducted on evaluation of effectiveness of polymer flooring system
compared with peel off mats highlights the pros and cons of each
system (Clibbon, 1970).
 Foot and Wheel Borne Contamination 373
Figure 1 Gown room with polymer flooring systems

Image courtesy of
TechTrak LLC

Polymer flooring systems are a semi-permanent installation (see

Figure 1 above). The mat is sealed to the existing flooring until
purposefully removed. Polymer flooring systems can be used
with most existing flooring including epoxy, vinyl, tile, and sealed
concrete.

Instead of adhesive, polymer flooring systems use Van der

Waal’s force theory of attraction. As two surfaces contact each other,
they form a bond. The surface of a shoe or wheel is attracted to the
mat. As the shoe or wheel leaves the mat, the debris on the shoe
or wheel adheres to the mat. Pliability of the mat ensures melding
around the shoe and wheel treads. This bond is only released with
water.

• Placement
Determining optimal placement of polymer flooring systems in
locations which see the most foot and wheel borne traffic depends
on risk analysis. These areas include gowning areas for human
traffic and access hallways for carts, forklifts, and other transport
from less clean areas (warehouses) to more controlled areas.
As shoe covers do not get compromised by polymer flooring
systems, they can be used outside and inside the gowning room,
as well as on both sides of the clean bench. Often companies opt
374 Cleanroom Contamination Prevention and Control

to eliminate the use of shoe covers after implementing polymer

flooring systems; however, this should depend on review of
long-term environmental monitoring trends. Utilizing polymer
flooring systems in key areas optimizes contamination removal
in the less controlled environments as well as contamination
prevention in critical areas.

• Size
To ensure proper use of polymer flooring systems, each mat
should be at least six feet long. Six feet allows for multiple
footfalls each.

In Figure 2, this polymer flooring system controls contamination

from an employee entrance using loose pile mats for gross
contamination control transitioning to the polymer flooring system
for fine particle control.

Figure 2 Correct placement of polymer flooring in corridor

Image courtesy of TechTrak LLC

 Foot and Wheel Borne Contamination 375

• Installation
Polymer flooring systems are not wall to wall carpeting. If a
hallway is six feet wide, a four-foot-wide installation that is at
least six feet long is sufficient. Personnel do not walk within a
foot of the wall. When using in doorways, spacing under the
doorway should be considered to accommodate the 1/24th
inch height of the mat with backing. If proper spacing is not
available, then placing the mat outside of the door swing is an
option. In this scenario personnel will take a step forward, grab
the door handle then step back to pull open the door. All these
steps count towards multiple footfalls.

Figure 3 Illustration of correct placement of polymer flooring

systems for doors with sweeps

Image courtesy of
TechTrak, LLC

The polymer flooring systems feature two thicknesses: one for human
and light cart traffic (1150 PSI), and a “tough” version for forklifts
(2000 PSI). When calculating the weight consider all four wheels and
the width of each. Care must be taken when choosing placement
for forklift traffic as turning on the polymer flooring system causes
the backing to separate from the reinforced double-sided flooring
adhesive leaving a bubble. Ideal areas include straight runs without
options for turning.
376 Cleanroom Contamination Prevention and Control
Figure 4 Ideal location for a tough polymer
flooring system installation

Image Courtesy of TechTrak LLC

Figure 4 shows an ideal location for a tough polymer flooring

system installation. Forklifts move over the mat in one direction,
unable to turn.

The placing of this tough installation controls both the employee

entrance as well as the roll up door for forklifts. Note the expansion
joint which is avoided to prevent a channel of water under the mat.
Expansion joints in concrete and cracks must be avoided or filled
with concrete sealer. Cleanouts or drain access must also be avoided.

• Cleaning
Squeegeeing or mopping the mat with a cleaning agent helps
remove the debris. With a short drying time, the cleaning
process is quick; and the mat is ready for reuse rapidly. Polymer
flooring is safe with all facility approved cleaning agents and can
be easily incorporated into the regular cleaning and disinfection
program. Floor buffer machines may be used with caution; only
 Foot and Wheel Borne Contamination 377

soft bristle brushes should be utilized; care must be taken not to

remove the cold weld seal from seams.

Polymer flooring that is longer than six feet should be cleaned

in four-foot sections from the clean side to dirty side. Cleaning
frequency should be based on traffic volume and the risk of
contamination ingress into the controlled or critical areas.

Figure 5 Saturated mat which needs cleaning

Image courtesy of TechTrak, LLC

• Color
Polymer flooring systems come in a variety of colors. All the
colors have the same effectiveness; the speckled versions “hide”
the appearance of dirt. If planned properly, color changes alert
technicians as they move into more controlled areas (see Figure
6). Speckled versions, used in dirtier areas, transition to flat colors
in cleaner areas. Flat gray shows less footprints than flat blue.
378 Cleanroom Contamination Prevention and Control
Figure 6 Speckled versus flat colors

Image courtesy of TechTrak, LLC

• Dirty and clean segregation

In smaller areas, such as gown rooms, colors can be cold welded
together to denote dirty side to clean side lines. The gowning
bench is placed on the flooring at the transition.

• Structural assembly
A polymer flooring system includes the mat, reinforced double
sided flooring adhesive, two-sided contact tape and the black
edging. Proper installation leaves a window box appearance.
The cold weld seal between the mat and edging ensures a
waterproof seal while the three stripes of contact tape provide a
waterproof edging to floor contact seal. Reinforced double sided
flooring adhesive ensures the center of mat remains in place.

To ensure proper seal, the floor type should be evaluated. For

example, if an epoxy flooring contains aggressive grit, it should be
sanded for adherence of the contact tape. All adhesives and remnants
from previous installations should be removed.
 Foot and Wheel Borne Contamination 379
Figure 7 Cold welding of two different color mats for gown room

Image courtesy of TechTrak, LLC

In conclusion, the goal of temporary or semi-permanent sticky mats

is to reduce contamination from entering the controlled and critical
areas. The effectiveness of these contamination control supplies
should be evaluated per ease of use and level of contamination
removed in conjunction with environmental monitoring trends.

REFERENCES
Clibbon, C. (1970) An Evaluation of the Effectiveness of Polymeric
Flooring Compared with “ Peel-off “ Mats to Reduce Wheel-and
Foot-Borne Contamination within Cleanroom Areas: Semantic
Scholar. Semantic Scholar. https://www.semanticscholar.org/paper/
An-evaluation-of-the-effectiveness-of-polymeric-%E2%80%9C-
%E2%80%9D-Clibbon/3feb8290bf680cb0fcc49264aafd99b9dcc60d25
380 Cleanroom Contamination Prevention and Control

Davis, C.P. (1996) “Normal Flora” in Medical Microbiology. 4th

edition. U.S. National Library of Medicine. https://www.ncbi.nlm.
nih.gov/books/NBK7617/

Estes, J.M., Hayes, Y.O., Freeman, Z.T., Fletcher, C.A., Baxter, V.K.
(2019) Effectiveness of Various Floor Contamination Control
Methods in Reducing Environmental Organic Load and
Maintaining Colony Health in Rodent Facilities. Journal of the
American Association for Laboratory Animal Science: JAALAS.
American Association for Laboratory Animal Science. https://
www.ncbi.nlm.nih.gov/pmc/articles/PMC6526484/

Sandle, T. (2012) Contamination Control Under Foot. Research &

Development World. https://www.rdworldonline.com/contamination-
control-under-foot/

Sandle, T. (1970) Examination of Air and Surface Particulate Levels

from Cleanroom Mats and Polymeric Flooring. European
Journal of Parenteral and Pharmaceutical Sciences. Euromed
Communications. https://www.research.manchester.ac.uk/portal/en/
publications/examination-of-air-and-surface-particulate-levels-from-
cleanroom-mats-and-polymeric-flooring(eba4b4d0-6188-45aa-b160-
7cc8c54b4008).html

Vieira, F.C.S., Nahas, E. (2005) “Comparison of Microbial Numbers

in Soils by Using Various Culture Media and Temperatures,”
in Microbiological Research. Urban & Fischer. https://www.
sciencedirect.com/science/article/pii/S0944501305000133
 Foot and Wheel Borne Contamination 381

ABOUT THE AUTHORS

Krishna Bell is the Vice President of Sales for the Midwest Region
at Acumen Technology, Inc. She has a bachelor’s degree in Biology
from University of Central Missouri. She has over 15 years of
experience in the healthcare sector. Her career spans from planning
clinical facilities to working in microbiological quality control and
testing at Merck Animal Health. This combination of clinical facilities
and pharmaceutical laboratory experience led her to understand
the value of contamination control and how contaminants can
affect animals and humans alike. She developed a passion for
contamination control which in turn help her to support companies
with contamination control products through Acumen Technologies.
She applies her varied experience in guiding customers develop
proactive contamination control plans though choosing effective
tools and equipment.

Ziva Abraham has over 35 years of academic, research, clinical and

industrial experience in Microbiology, and Quality Assurance. Ziva
has received her master’s degree in Microbiology and has conducted
research on developing Microbial Insecticides using entomogenous
bacteria and fungi towards her Ph.D. degree.

She worked as a clinical scientist for many years and established

clinical laboratory systems in Israel. In this capacity Ziva evaluated
the first automated microbial identification system, introduced
new technologies for HIV testing, and many rapid testing methods
into the group of laboratories she helped establish and manage
for Maccabi Medical. These laboratories conducted clinical
testing in microbiology, chemistry, parasitology, hematology and
immunology.

She is a passionate microbiologist and mycologist and provides

training worldwide on various microbiology and contamination
control topics. Her clinical experience helps her evaluate and
teach the clinical implications of microbial contamination on
patients. Ziva is passionate about fungi; as in her research she had
382 Cleanroom Contamination Prevention and Control

collected, identified, and tested hundreds of species. She routinely

teaches hands-on fungal identification as well as investigation
and remediation of mold contamination and most importantly the
emergence of new fungal infections.

Ziva established Microrite, a California based consulting firm,

in 1998 as an independent microbiology consultant. Ziva used her
extensive research experience with fungi and clinical organisms
and their impact on human health to build an internationally
renowned contamination control team of experts. Her team consists
of facilities, cleanroom, airflow, static charge, validation, quality and
microbiology professionals with practical knowledge in their field
and involvement in industry standards and guidance organizations.
Ziva’s team works congruently to resolve client issues, prevent
contamination through design and help clients produce safe
products for human and veterinary use.
 13

VALIDATION OF DISINFECTANTS

Tim Sandle
Pharmaceutical Microbiology Interest Group (Pharmig), UK
Laura Guardi
Pharmaceutical Microbiology Interest Group (Pharmig), UK
Rachel Kirkham
Ecolab Life Sciences, Northwich, UK
Kim Morwood
MGS Laboratories Ltd, Portsmouth, UK

INTRODUCTION
In most countries there is a legal requirement to prove that
disinfectants for sale are both safe and effective and this is achieved
through testing and registration with the appropriate authority. For
example: The Biocidal Products Regulation in the European Union
(EU) and the Federal Insecticide, Fungicide, and Rodenticide Act
(FIFRA) in the US, set out the necessary requirements. Although
this testing can provide the end user with a degree of assurance
regarding the product performance, the test methods mandated for
registration do not reflect how the disinfectant would be used in the
unique setting of a pharmaceutical cleanroom.

383
384 Cleanroom Contamination Prevention and Control

There are differences between US and European requirements

for the qualification of disinfectants, which are clearly laid out in
the chapter. The reader should determine which set of regulations
is applicable for the territory within which he or she operates and
follow the advice in this chapter accordingly.

Where disinfectants are used in a Good Manufacturing Practice

(GMP) environment it is a regulatory expectation that:

▪ Testing is performed to demonstrate that they are fit for

purpose, i.e., that they provide a sufficient level of reduction in
viability for the types of organisms expected to be encountered
on the surfaces on which they will be used (PIC/S PI 007-6; FDA
Guidance for Aseptic Processing).

▪ Where microbial monitoring of surfaces in clean rooms is

carried out, validation should be performed on the test method
to confirm that sanitizing agents do not influence the recovery of
microorganisms (EU GMP Annex 15).

In this chapter the following topics are discussed:

▪ The most common methods used to prove disinfectant efficacy
and selection of test parameters.
▪ Validation of environmental monitoring contact plates.
▪ Requirements for re-validation.
▪ Considerations for qualification of disinfectants used for hand
sanitization.
In doing so the chapter presents the broad global requirements for
disinfectant validation.
 Validation of Disinfectants 385

DEFINING A DISINFECTANT VALIDATION STRATEGY

AND SELECTION OF TEST PARAMETERS
As the introduction indicates, approaching disinfection validation
is a multi-stage process and it is important to develop a clear
strategy, ideally in the form of a protocol or master plan. Beyond
the requirements stated in the introduction, the regulations do
not provide any detail regarding strategies for qualification of a
cleaning and disinfection regime and laboratory test methods,
other than requiring that qualification is conducted and expressing
a preference for the surface test (as indicated in the FDA Aseptic
Processing Guidance (2004) and the 2020 draft version of EU GMP
Annex 1. Furthermore, USP <1072> Antiseptics and Disinfectants
(a non-binding General Chapter) provides some useful theoretical
and practical guidance regarding the use of disinfectants in GMP
environments, including recommendations for qualification and
testing. ASTM (American Society for Testing and Materials) E2614
Standard Guide for Evaluation of Cleanroom Disinfectants is also a
useful source of information.

The following stages should be considered as part of a validation

strategy:

• Disinfectant use-dilution/suspension tests.

• Disinfectant surface challenge tests.
• Validation of the neutralizing effect of contact plates.
• Qualification of hold times for diluted disinfectants.
• Field trial of cleaning and disinfection regime.

It is incumbent on companies to provide a reasoned and justified

approach to their disinfectant validation studies. A quality risk
management approach should be applied. It may not be appropriate
to perform all of the above steps and the extent of testing that is
performed will be dependent on where and how the disinfectant
is used. For this reason, it may be useful to document the overall
strategy in the form of a Validation Master Plan.
386 Cleanroom Contamination Prevention and Control

USE-DILUTION AND SUSPENSION TESTS

Where a large number of disinfectants at various concentrations
and/or contact times are being assessed, use-dilution or suspension
methods may be useful as screening tests to pre-select the most
appropriate disinfectant/concentration/contact time combination for
subsequent surface testing. Such testing usually takes the form of
quantitative suspension tests (e.g., tests based on EN 1650:2019; EN
1276:2019; EN 13704:2018), whereby a known number of organisms is
mixed in solution with interfering substance and the test disinfectant
for a set contact time and, following neutralization of the disinfectant,
the number of organisms inactivated is calculated. Suspension tests
are relatively simple to carry out and can identify products most
likely to perform well under specific conditions. However, they do not
necessarily predict how the disinfectant will perform on contaminated
surfaces. It should also be noted that, when using EN 1650, EN 1276
and EN 13704, as the disinfectant is tested in solution, the actual con­
centration of a ready-to-use disinfectant in the test is 80% of its full
strength. However, it is possible to increase the concentration of the
inoculum and interfering substance and reduce the volume used to
counteract this. EN 13727:2012; A2:2015, a method intended for test­
ing of disinfectants used in medical areas, has been devised so that
ready-to-use disinfectants can be tested at 97% concentration. It is
likely that future revisions of EN 1650, EN 1276 and EN 13704 will
likewise be revised to permit testing of ready to use disinfectants at
97% concentration. Suspension tests are likely to be of limited value,
particularly where only a small selection of concentrated disinfectants
and/or ready to use disinfectants are to be evaluated.

DISINFECTANT SURFACE CHALLENGE TESTS

This is the most common type of testing performed for the purpose
of validation of cleanroom disinfectants and involves application of
a known number of organisms mixed with an interfering substance
onto a hard, non-porous surface. After the inoculum has dried, the
disinfectant is then applied for a set contact time and, following
neutralization of the disinfectant and recovery from the surface, the
 Validation of Disinfectants 387

number of organisms inactivated is calculated. There are a variety of

internationally recognized test methods available: both qualitative
and quantitative, with and without mechanical action. The reduction
of the number of test organisms caused by a disinfectant is generally
expressed as decimal logarithm (log), commonly referred to as
the log reduction. In order to demonstrate compliance with pre-
determined acceptance criteria specified in terms of a log reduction,
it is necessary to use a quantitative test method. The incorporation
of mechanical action into the method is significant as disinfectants
used in the cleanroom will usually be applied using some form of
mopping or wiping. Test methods that do not incorporate mechanical
action could therefore be considered as “worst case”.

The Association of Analytical Communities (AOAC)

International Germicidal Spray Test provides a qualitative endpoint
(presence or absence) and as such, is not practical for demonstrating
compliance to a specified log reduction. The semi-quantitative
AOAC Use Dilution Tests (955.14; 955.15; 964.02) and sporicidal
hard surface test (966.04) involve immersion of the test surface
into the disinfectant and require complete kill of the test inoculum,
without any mechanical action and as a result, may be of limited
use in demonstrating efficacy against the low level of organisms
expected in a cleanroom. These methods also have the disadvantage
of requiring a high number of replicates, which in turn requires a
high degree of skill from the analyst in performing the test in order
to manipulate multiple surfaces with precision timing.

The ASTM hard surface methods E2111 and E2197 and EN

13697:2015; A1:2019 are quantitative methods, making it possible to
produce statistically valid data using many fewer test and control
carriers than the AOAC methods described above which are based
on most probable numbers. However, the E2111 method is of limited
use for validation of cleanroom disinfectants as the test surface is
the inside of a glass vial, limiting its applicability to testing various
cleanroom surfaces. As the E2111 and E2197 methods are not
published by ASTM, they do not specify acceptance criteria, simply
the method to follow. In addition to lacking a defined performance
388 Cleanroom Contamination Prevention and Control

standard, these tests do not incorporate any mechanical action,

limiting their applicability to the cleanroom environment.

However, EN 16615:2015, for assessment of disinfectants used in

the medical area, is both quantitative and incorporates mechanical
action by means of using a wipe to apply the disinfectant to a series
of test fields and may be adapted to test biocidal efficacy on a variety
of hard, non-porous surfaces. As such, this method has become a
popular choice for validation of cleanroom disinfectants.

MODIFICATION OF TEST PARAMETERS

As discussed above, the standard test methods used for disinfectant
registration do not accurately reflect how the disinfectant would be
used in practice in a cleanroom setting. The regulatory authorities do
not demand compliance with the standard test methods described
above, although it would appear that validation studies based on
published methods are likely to be accepted as adequate proof of
disinfection efficacy, provided of course that they are relevant to the
processes in question. The main advantage of using an internationally
recognized standard test is that the method is usually self-validating
due to the inclusion of appropriate system suitability checks and
controls, therefore eliminating the need for in-house method
validation.

Individual companies need to consider the available methods

and how they may be modified in order to meet their individual
requirements. A summary of the most common EN, AOAC and
ASTM methods is provided at the end of this section. The following
test parameters should be considered when designing a study:
▪ Test surfaces.
▪ Test organisms.
▪ Contact time.
▪ Acceptance criteria.
 Validation of Disinfectants 389

▪ Water quality.
▪ Temperature.
▪ Soiling.
▪ Neutralizers.
It should be noted that when an organization chooses to use the EN,
AOAC or ASTM test methods to validate its disinfection procedures,
care must be taken in making a claim of compliance with the
standard. In order for a claim to be made, full compliance with all
the obligatory requirements stated in that standard must be met. If,
however, the method is modified to make it more applicable to a
particular situation and the obligatory test conditions have not been
included, then a claim of compliance to the standard cannot be made.

With many of the test methods of CEN Technical Committee 21,

ring trials have been performed to determine their feasibility and to
evaluate their level of reproducibility. The results and the statistical
analyses of the ring trials are summarized in each of those standards,
including ensuing recommendations. Modifying the test methods
may more accurately reflect actual usage conditions but may also
adversely affect test reproducibility.

Notwithstanding this concern, in some instances it may be

more appropriate to modify the test procedures to make them more
relevant to particular applications rather than rigidly applying
official methods that may not be entirely relevant. EN 14885:2018,
which describes the application of the EN standard methods,
provides further guidance with regards to best practice for the
control of imprecision.

Disinfectant efficacy testing will not be a routine activity for

most pharmaceutical microbiology laboratories. The methods used
are technique sensitive and lack of experience can further increase
imprecision. For this reason, it is common practice to use a specialist
contract testing laboratory for these studies.
390 Cleanroom Contamination Prevention and Control

Test surfaces
EN and AOAC surface test methods specify the use of stainless-
steel or glass coupons, stainless steel or porcelain cylinders, and
in the case of the AOAC, sporicidal hard surface test, silk suture
loops. While stainless steel is often found in abundance in GMP
areas, there are also likely to be other surface types that are more
predominant (e.g., floor and wall coverings) and more challenging
to clean and disinfect due to their uneven surface finish (e.g., vinyl
and glass). The FDA Guidance for Aseptic Processing, USP <1072>
and PIC/S PI 007-6 recommend including samples of surfaces from
the cleanroom environment in the test panel. The type of surfaces
to be included in the study should be selected according to risk
assessment considering:

▪ Predominance – total surface area.

▪ Surface finish – how easy/difficult are they to clean/disinfect.

▪ Criticality – proximity to critical process steps, potential for

indirect product contact.

▪ Suitability – the surface must be non-porous and possible to cut

into coupons of a suitable size for testing.

▪ Availability – in older facilities it may not be possible to obtain

sufficient samples of the surface for testing.

The particular surface(s) upon which a disinfectant is intended to

be used should also be considered. For example, 70% Isopropyl
Alcohol (IPA) intended for use on gloved hands and work surfaces
would not require testing on floor or wall materials.

Test organisms
The EN and AOAC methods use characterized strains from recog­
nized culture collections (e.g., ATCC, NCTC); whereas the FDA
Guidance for Aseptics Processing, USP <1072>, and other regulat­
ory authorities’ expectations are to include facility environmental
 Validation of Disinfectants 391

isolates in the test panel. Including facility isolates in the test

panel can provide valuable additional information regarding their
susceptibility to the disinfectants in use. The selection of organisms
for testing should be made according to risk assessment considering:

▪ Manufacturer claims for biocidal activity – e.g., it is not appro-

priate to test a non-sporicidal product using a spore suspension.

▪ The spectrum of organisms that could reasonably be expected to

occur in the cleanroom – i.e., Gram-positive cocci, spore-forming
bacilli, Gram-positive non-spore forming rod, yeast, mold.

▪ A historical review of environmental monitoring data – each

facility will have unique microbial flora with only a few species
representing the majority of organisms recovered from the
environment.

Where a certain type of organism is not available as a facility

isolate (e.g., mold cultures that are relevant but cannot be routinely
recovered), it is acceptable to substitute with a type strain from a
culture collection.

Testing of disinfectants for use in a brand new facility is uniquely

challenging in that there are likely to be no, or limited, historical
data and/or available environmental isolates for culture. In such
circumstances, it may be appropriate to perform limited testing
using organisms from culture collections that are representative of
the expected cleanroom flora as a proof-of-principle prior to routine
use, followed by more extensive testing once sufficient data become
available.

The way in which cultures are prepared for testing is of

paramount importance. To ensure maximum resistance and
reproducibility EN and AOAC methods use vegetative cultures in
the exponential phase of growth and spore suspensions prepared
according to specific methods. For example: the use of cultures of
the more resistant “spiny” spores of A. brasiliensis in EN 1650: 2019
and EN 13697:2015; A1: 2019; thorough washing of B. subtilis spore
392 Cleanroom Contamination Prevention and Control

suspensions and checking for the absence of vegetative cells in EN

13704:2018.

Therefore, wherever possible, it is advised that the culture

methods described in the standard tests are adopted.

Contact times
The contact time is defined as the total time an organism is
exposed to the antimicrobial action of a disinfectant. The contact
time necessary to achieve a desired level of reduction in microbial
viability may differ according to the target organism. For example,
longer contact times are typically required for fungicidal activity
than bactericidal activity; inactivation of bacterial spores typically
requires longer contact times than those required for fungicidal
activity. A summary of the standard contact times for EN and AOAC
methods is provided in Table 1. Some of these contact times are far
longer than that which would be practically useful in a GMP setting
(e.g., a maximum 60-minute contact time for sporicidal activity in
EN 13704:2018). The aim should be to validate the shortest possible
contact time needed to prove biocidal efficacy (i.e., meet acceptance
test criteria). Consideration should be given to the increased rate of
evaporation when using a volatile disinfectant, such as alcohol. As the
test acceptance criteria could reasonably be reduced by comparison
to those stipulated in the EN and AOAC methods, it is often possible
to validate a more appropriate contact time. More rapid evaporation
of the disinfectant may occur on warm surfaces or where the treated
surface is subject to low humidity or high airflow conditions, as is
sometimes found in cleanroom operations. Typical surface drying
times in a cleanroom are in the order of 5–8 minutes. Historically
there have been concerns with regards to contact times that result in
a dry surface in practice (i.e., the surface is not wet for the full contact
time). It is regulatory expectation that the wet contact time applied
in practice reflects the contact time applied during validation. The
purpose of in vitro testing is to determine appropriate parameters to
be applied practice; the final stage of validation is the in situ, or field
testing. Therefore, the success of the field trial demonstrates that the
 Validation of Disinfectants 393

regime is fit for purpose. Ongoing assessment of the effectiveness of

the regime is demonstrated through the environmental monitoring
program.

Acceptance criteria
The reduction of the number of test organisms caused by a
disinfectant is generally expressed as decimal logarithm (log) and
is commonly referred to as the log reduction. In the EN standard
methods, this is calculated by comparing the number of survivors
from the test article to the number of survivors in a control (test
performed simultaneously without the disinfectant) in order to take
into account any reduction in viability that has occurred as a result
of the testing process itself. There should be relatively little reduction
in viability observed in the control. As the level of reduction in
viability is expressed logarithmically, when interpreting results, it is
necessary to take into account the concentration of starting inoculum
for suspension tests, or the number of organisms surviving on the
control surfaces for surface test. To ensure reproducibility between
tests, careful attention should be paid to obtaining a consistent
concentration of starting inoculum. The starting inocula and
minimum log reductions required to satisfy the EN, AOAC, and
ASTM methods are summarized in Table 1.

USP <1072> proposes acceptance criteria which are lower than

those specified by the standards, due to the unique circumstances of
the cleanroom setting:

▪ Disinfectants could be less effective against the high numbers

of organisms used during testing than against the low numbers
expected in a cleanroom.

▪ Stressed environmental organisms may be more susceptible to

disinfectants than the cultures using in testing which are in the
exponential phase of growth.
394 Cleanroom Contamination Prevention and Control
Table 1 Summary of starting inoculum and acceptance criteria

Method Test organisms Starting inoculum Acceptance criteria

EN 1276:2019 Staphylococcus 1.5 × 10 to
8
5 log reduction,
(bactericidal aureus; Enterococcus 5.0 × 108 CFU/mL temperature
suspension test) hirae; Pseudomonas 4–60⁰C in 1–60
aeruginosa; minutes
Escherichia coli
Enterococcus faecium
for above 40⁰C)
EN 1650:2019 Candida albicans; 1.5 × 107 to 4 log reduction
(yeasticidal Aspergillus brasiliensis 5.0 × 107 CFU/mL temperature
and fungicidal 4–40⁰C in 1–60
suspension test) minutes
EN 13704:2018 Bacillus subtilis; 1.5 × 106 3 log reduction
(sporicidal Bacillus cereus to 5.0 × 106 CFU/mL temperature
suspension test) (optional) 4–75⁰C in 1–60
minutes
EN 13697:2015; Staphylococcus aureus; Bacteria:; 1.5 × 108 to Bacteria: 4 log
A1:2019 Enterococcus hirae; 5.0 × 108 CFU/mL reduction Yeast
(bactericidal, Pseudomonas P. aeruginosa: 1.5 × 109 and fungal spores:
yeasticidal, and aeruginosa; to 5.0 × 109 CFU/mL 3 log reduction
fungicidal surface Escherichia coli; (clean conditions) temperature
test) Candida albicans; P. aeruginosa: 1.5 × 108 4–40⁰C in 1–60
Aspergillus brasiliensis to 5.0 × 108 CFU/mL minutes
(dirty conditions)
Fungal spores: 1.5 × 107
to 5.0 × 107 CFU/mL
Yeast 1.5 × 108 to 5.0 × 108
CFU/mL (clean conditions)
1.5 × 107 to 5.0 × 107
CFU/mL (dirty conditions)
EN 16615:2015 Staphylococcus aureus; Bacteria: Bacteria: 5 log
(bactericidal and Enterococcus hirae; 1.5 × 109 to 5.0 × 109 CFU/ reduction in test
yeasticidal surface Pseudomonas mL (6.88 – 8.40 log [7.9 × field 1 + mean CFU
test with mechanical aeruginosa; 106 – 2.5 × 108] CFU on on test fields 2 to
action) Candida albicans drying control at start and 4 of <50 in 1–60
end of contact time) minutes
Yeast and fungal spores: Yeast and fungal
1.5 × 108 to 5.0 × 108 CFU/ spores: 4 log
mL (5.88 – 7.40 log [7.9 × reduction in test
105 – 2.5 × 107] CFU on field 1 + mean CFU
drying control at start and on test fields 2 to
end of contact time) 4 of <50 in 1–60
minutes
 Validation of Disinfectants 395

Method Test organisms Starting inoculum Acceptance criteria

ASTM E2111‑2 Staphylococcus aureus; N/A** N/A*(**)
Carrier Test Pseudomonas.
(bactericidal, aeruginosa;
fungicidal, Trichophyton
mycobactericidal, mentagrophytes;
sporicidal*) Candida albicans;
Aspergillus brasiliensis;
Mycobacterium terrae;
Bacillus subtilis;
Clostridium sporogenes
ASTM E2197 Staphylococcus aureus; N/A** N/A**
(Bactericidal, Pseudomonas.
fungicidal, aeruginosa;
mycobactericidal, Trichophyton
sporicidal, mentagrophytes;
virucidal*) Candida albicans;
Aspergillus brasiliensis;
Mycobacterium terrae;
Bacillus subtilis;
Clostridium sporogenes;
Human Adenovirus 5;
Hepatitis A Virus
Strain HM-175;
Canine Parvovirus
– Strain Cornell
780916–80;
Feline calicivirus F9;
Human Rhinovirus 37
or 14;
Human Rotavirus
strain Wa;
Murine Norovirus

* The organisms listed are those recommended in the method, other organisms may also be used.

** This is method is not a standard and so does not include acceptance criteria. The method is
designed to have survivors and advises that in order to allow proper statistical evaluation of results,
the size of the test inoculum should be sufficiently large to take into account both the performance
standard and the experimental variation in the results.

▪ In reality, disinfectants are applied by wiping/mopping which

enhances bioburden reduction by physical removal or organisms
from the test surface in addition to reduction in microbial
viability from the disinfectant
396 Cleanroom Contamination Prevention and Control

The acceptance criteria proposed by USP <1072> are:

▪ Vegetative bacteria – 3 log reduction.

▪ Bacterial spores – 2 log reduction.

Unfortunately, USP <1072> does not suggest starting inoculum

concentration or indicate acceptable log reduction values for yeast
or mold spores. It should also be noted that, in contradiction to the
assumption that stressed environmental organisms are likely to
be more susceptible to the activity of disinfectants, in some cases
environmental stresses such as temperature, nutrient limitation, and
exposure to reactive oxygen species, are known to reduce bacterial
susceptibility to a variety of antimicrobials, including disinfectants,
through the initiation of stress response pathways.

The recently issued PDA Technical Report #70 (Fundamentals

of Cleaning and Disinfection Programs for Aseptic Manufacturing
Facilities) recommends >1 log reduction for non-spore-formers,
mycoplasma, mold spores and bacteria spores. However, to date
this approach has not been endorsed by any regulatory authority
and is not recommended by the authors.

Reference to the seemingly conflicting documents discussed

above can be of use when setting acceptance criteria for disinfectant
validation studies. The following aspects should be taken into
account:

▪ That different types of organisms (e.g., vegetative bacteria,

yeasts, mold and bacteria spores) have differing degrees of
susceptibility to disinfectants.

▪ The classification (and hence maximum permitted microbial

limits) of the area(s) where the disinfectant will be used.
For example, refer to Annex 1 of EU GMP and USP <1116>
Microbio­logical Control and Monitoring of Aseptic Processing
Environments.
 Validation of Disinfectants 397

▪ The level of bioburden typically recovered from the area

(remembering that the limitations of the culture method
will mean that it may not be possible to culture and count all
organisms that are actually present).

▪ Whether or not the test method incorporates any mechanical

action which may enhance microbial reduction.

It is good practice to document the rationale that was used to

determine appropriate acceptance criteria.

Water quality
The level of mineral content (i.e., the hardness of water) has an
impact on disinfectant efficacy; disinfectants diluted with hard
water will typically have reduced efficacy compared to the same
disinfectant prepared with soft or purified water. For this reason,
the EN methods use “water of standard hardness” for dilution of
disinfectants that are supplied as a concentrate and diluted prior
to use. In a GMP environment disinfectant solutions are typically
prepared using purified water or water for injection, both of which
contain very low levels of dissolved minerals. Therefore, the water
that will be used in practice should also be used in vitro.

Temperature
The efficacy of disinfectants can be temperature dependent; the same
concentration of disinfectant may reduce the number of organisms
more rapidly at as temperature increases, and more slowly as
temperature falls. Therefore, in order to achieve the same level of
reduction in microbial viability, a longer contact time may be needed
when a disinfectant is used at low temperature (e.g., in a cold room or
refrigerator) than at room temperature. The standard EN test methods
have historically been conducted at obligatory conditions of 20°C (+/-
1°C), whilst this remains a commonly applied test temperature, more
recent revisions of these standards have replaced their obligatory
398 Cleanroom Contamination Prevention and Control

conditions with wider temperature ranges, for example EN 13697

applies a test temperature range of 4°C to 40°C. The impact of
temperature on biocidal efficacy should be taken into account when
defining a testing regime. A summary of the standard temperature
ranges for EN and AOAC methods is provided in Table 1.

Soiling
Just as the presence of inorganic material can affect biocidal activity,
organic matter can also reduce the efficacy of a disinfectant. The
standard methods make allowances for this and provide for
testing under “dirty” and “clean” conditions by the addition of
specified soiling agents, known as interfering substances, at specific
concentrations. The interfering agent used is selected according to
application, for example the interfering agent of choice for GMP
purposes is bovine serum albumin (BSA). USP <1072> recommends
testing under “clean” conditions as this is most representative of the
levels of soiling expected in a cleanroom.

It should be noted that “clean” conditions do not indicate the

absence of an interfering agent, as a small quantity is required to
protect the inoculum during drying. Historically there has been an
issue with viability of Gram negative organisms and yeast when
testing according to EN 13697, as these organisms are very sensitive
to drying. In an attempt to improve viability, the latest edition of the
EN 13697 standard uses a higher starting inoculum for P. aeruginosa
and C. albicans with BSA.

There is a significant difference in the amount of interfering

substance used to simulate “dirty” conditions in the EN and AOAC
methods. Concentrations of BSA in the test mixture are 0.3% in EN
methods and 5% in AOAC methods. This reflects the fact that the
test conditions in the AOAC methods are used for assessment of the
performance of “one-step cleaner disinfectants” (i.e., formulations
which include a detergent); whereas the EN methods are used for
assessment of disinfectants without any cleaning activity.
 Validation of Disinfectants 399

Neutralizers
The use of an appropriate neutralizer is critical to ensuring that the
efficacy within the specified contact time is being assessed. The type
of neutralizer used will depend on the type of disinfectant being
tested. The method design should include controls to demonstrate
that:

▪ The test method does not have a deleterious effect on microbial

recovery (i.e., microorganisms are not inactivated as a result of
the test method, rather than due to exposure to the disinfectant).

▪ The neutralizer does not have a deleterious effect on microbial

recovery (i.e., microorganisms are not inactivated as a result of
exposure to the neutralizer, rather than due to exposure to the
disinfectant).

▪ Neutralizer used is capable of quenching the activity of the

disinfectant (i.e., that neutralization is occurring immediately
after the contact time).

Without these controls it is impossible to determine that microbial

reduction is due to the disinfectant.

VALIDATION OF THE NEUTRALIZING

EFFECT OF CONTACT PLATES
As mentioned at the beginning of the chapter, the latest revision of
Annex 15 of EU GMP had a requirement to ensure that sanitizing
agents used on cleanroom surfaces do not negatively affect the
recovery of microorganisms (EU GMP Annex 15). The majority of
disinfectants will leave some non-volatile residues on a surface after
drying; the amount of residue remaining varies depending on the
active and product formulation. When contact plates are applied to the
surface, these disinfectant residues will be transferred to the agar and
will be re-solubilized which may inhibit recovery of organisms. The
use of contact plates containing appropriate neutralizers can prevent
400 Cleanroom Contamination Prevention and Control

this phenomenon. It is therefore appropriate to perform testing to

prove that low numbers of organisms can be recovered in the presence
of these residues. As with disinfectant efficacy testing, facility isolates
should be included in the test panel. A wide variety of microbiological
media formulated with neutralizing agents are commercially available,
suitable for use with many different types of disinfectant.

QUALIFICATION OF HOLD TIMES

FOR DILUTED DISINFECTANTS
Many disinfectants become unstable when diluted. Where
disinfectants are prepared in bulk from concentrate and held for
prolonged periods prior to use (e.g., longer than one work session)
it is advisable to qualify the hold time. Testing should be performed
to prove that the solution remains stable and efficacious until the
end of the storage period. The potential for microbial contamination
of the solution should also be considered; bioburden in non-sterile
disinfectants should not significantly increase during storage, and
sterile solutions should remain sterile throughout the hold time.
Where bioburden testing is performed it is important to use an
effective neutralizer to ensure maximum microbial recovery, as
discussed previously.

FIELD TRIALS
Whilst in vitro testing can cover some practical usage conditions, it
does not take into account all factors that may influence the success
of the cleaning and disinfection regime as a whole. Such factors
may include methods of application, frequency of disinfection, and
rotational patterns followed. As such, a field trail (also known as in
situ testing) should be performed. No standard has been published
for field trials, though some guidance is provided in Annex C of EN
14885:2018.

Typically, this involves increased environmental monitoring,

prior to and sometime after incorporation of new disinfectant
 Validation of Disinfectants 401

into the hygiene regime. Assessment would be based on analysis

of the trends of microbial counts and the types of microorganisms
recovered.

RE-VALIDATION
Just as GMP equipment, facilities, utilities and systems should be
evaluated at an appropriate frequency to confirm that they remain
in a state of control, the cleaning and disinfection regime should
also be periodically assessed. It is good practice to consider the
need for additional validation studies as part of the periodic (e.g.,
annual) review of environmental monitoring trends. For example,
if a new predominant organism is isolated that was not included in
the original test panel, it may be appropriate to perform additional
laboratory testing to ensure that the organism is susceptible to the
disinfectants in use.

Any planned changes that may affect the performance of the

hygiene regime should be formally documented and the impact on
the validated status assessed using the change management process.
For example, change to a disinfectant formulation could impact its
efficacy or refurbishment of the facility could result in a new material
that should be subjected to surface challenge testing. Re-validation
need not involve the full scope of validation, but the critical aspects
based on risk (e.g., repeating a field trial or running surface efficacy
tests using environmental isolates).

CONSIDERATIONS FOR QUALIFICATION

OF HAND HYGIENE PRODUCTS
The CEN Technical Committee 216 have issued two test methods
for assessment of products designed as antiseptic handwashes (i.e.,
with the addition of water) or as antiseptic handrubs (i.e., without
the addition of water), preparations which are directed against
transient skin microorganisms.
402 Cleanroom Contamination Prevention and Control

▪ EN 1499: 2013 – Phase 2 / Step 2 Hygienic Handwash.

▪ EN 1500: 2013 - Phase 2 / Step 2 Hygienic Handrub.
The requirement of the tests is to confirm that the product in
question produces a significant reduction of the test organism (an
attenuated strain of Escherichia coli) when compared to a reference
handwash (un-medicated liquid soap) or when compared to a
reference handrub (60% v/v propan-2-ol). The tests are performed
using a panel of 18–22 volunteers who perform the test using the
handwash/handrub under test and then repeat the exercise using
the control reference handwash/handrub in a cross-over fashion.

This test can safely be performed only when using the specified
attenuated strain of E. coli, therefore it is not possible to produce
facility-specific data by repeating the test using in-house isolates on
human volunteers. EN 1499 and EN 1500 testing is only of value
where data regarding the efficacy of the handwash/handrub is not
available from the manufacturer. If efficacy data for these standards
are available and the product is used in practice according to the
conditions stipulated by the manufacturer (in terms of volume
applied and contact time) there is limited value in repeating this
testing.

SUMMARY OF COMMON TEST METHODS

There is no global, unified approach to the validation of disinfectants.
The two primary approaches are the European and US standards.
Summaries of both approaches are presented here. The user should
adopt the one appropriate to where their manufacturing facility is
located.

TEST METHODS FROM CEN

The European Committee for Standardization (CEN, Comité
Européen de Normalisation) set up a technical committee (Technical
Committee 216) to produce harmonized test methods for assessment
 Validation of Disinfectants 403

of the efficacy of disinfectants and antiseptics. These standards

include a mixture of suspension tests and surface tests to cover
bactericidal, fungicidal, viricidal, sporicidal and mycobactericidal
activity. The philosophy behind the CEN standards is to provide
initial data to demonstrate that the disinfectant under test has
antimicrobial activity, which can then be used to establish effective­
ness under in-use conditions. Data from these tests are used in the
registration process of products for sale in the EU, as per the Biocidal
Products Directive.

EN 1040: 2005 Phase 1 – Basic Bactericidal Activity;

EN 1275: 2005 Phase 1 – Basic Fungicidal Activity
These tests are limited in scope and are intended as suspension tests
for use during product development to confirm whether a particular
compound has bactericidal/fungicidal activity under the defined
test conditions; they are not designed to evaluate a product for
any specific intended use and may not be used to support product
claims for registration. These tests are therefore not appropriate for
evaluation of disinfectants for use in pharmaceutical cleanrooms.

EN 1276: 2019 Phase 2/Step 1

– Quantitative Bactericidal Suspension Test
This is a more comprehensive test involving at least two Gram-
positive bacteria (Staphylococcus aureus and Enterococcus hirae) and
at least two Gram-negative organisms (Pseudomonas aeruginosa and
Escherichia coli) with the provision for additional test organisms if
desired. For tests temperatures exceeding 40°C, Enterococcus faecium
is also tested. Incorporated into the test procedure are interfering
substances to simulate clean and dirty conditions using different
levels of BSA and the effect of water hardness. Temperatures
and contact times can be incorporated based on manufacturer’s
recommendations and are further detailed in Table 1.
404 Cleanroom Contamination Prevention and Control

Three disinfectant concentrations are included to cover the

active and non-active range; this is to provide some control on the
test method (i.e., the non-active concentration would be expected to
fail the test). There are also additional controls to ensure that the test
procedures are satisfactory, to demonstrate that the neutralization
procedure is effective and to ensure that the neutralization agents do
not themselves inactivate the test organisms.

The basic requirement is at least a 5-log reduction of all the test

organisms. For hand wash products, a 3-log reduction is required.

This test is intended to take account of some of the environmental

parameters (e.g., water hardness, organic load) as well as the
effect of the contact time, all of which may influence the efficacy
of the disinfectant under in-use conditions. However, it should
be noted that ready-to-use disinfectants will be disadvantaged in
comparison to concentrated disinfectants when tested according to
this method, as some dilution is always produced by the addition
of inoculum and interfering substance. Ready to use products can
only be tested at a concentration of 97% or less.

EN 1650: 2019 Phase 2/Step 1

– Quantitative Fungicidal Suspension Test
This test procedure serves a similar function to the Quantitative
Bacterial Suspension Test (EN 1276) but is intended to cover
fungicidal activity.

This test utilizes the same two organisms as used in the Phase
1 Basic Fungicidal Test (Candida albicans and Aspergillus brasiliensis)
with the provision for additional test organisms if desired. It in­
corporates the same interfering substances and test controls as in the
Quantitative Bactericidal Suspension Test and has the same limit­
ation regarding testing of ready to use products. The temperature
and contact time are recommended by the disinfectant manufacturer.
 Validation of Disinfectants 405

The basic requirement is at least a 4-log reduction for all the

test organisms. This requirement is less stringent than that required
for the quantitative bacterial suspension test and reflects the
expected reduced susceptibility of fungi to disinfectant preparations
compared to bacteria.

EN 13704: 2018 Phase 2/Step 1

– Quantitative Sporicidal Suspension Test
This test is a quantitative suspension test for establishing whether a
chemical disinfectant has or does not have sporicidal activity.

It incorporates the same principle, test controls, temperature

and interfering substance as the other suspension tests. Other key
differences include the use of a spore forming organism that is tested
as a spore suspension. The methods include a check of the suspension
to ensure the absence of any vegetative cells, and also checks for
the susceptibility during the shelf life of the spore suspension.
This method has the same limitation as the other suspension tests
regarding ready to use disinfectants.

To meet the requirements of this test, the product must be

capable of producing at least a 3-log reduction in the number of
bacterial spores belonging to the reference strain of Bacillus subtilis
ATCC 6633 (with the additional option of Bacillus cereus for certain
disinfectant product types). Temperature and contact time to be
recommended by the disinfectant manufacturer.

EN 13697: 2015; A1: 2019 – Phase 2/Step 2

– Quantitative Non-Porous Surface Test
This test is similar to the Quantitative Suspension Tests but is meant
to assess the efficacy of a product against organisms dried onto a
surface. As it stands it combines tests for both bactericidal and
fungicidal surface activity and incorporates the same test organisms
as used in the Phase 2 Step 1 Quantitative Bactericidal and Fungicidal
406 Cleanroom Contamination Prevention and Control

Suspension Tests. It also utilizes the same interfering substances,

contact time and temperature.

This test involves organisms and interfering substance being

dried onto the surface of stainless-steel coupons. After application
of the disinfectant and the required contact time, the product is
neutralized, and the remaining organisms are removed from the
surface (by mechanical action using glass beads) and enumerated
using the pour plate or spread plate method.

The minimum biocidal activity to meet the requirements of this

test, a 4-log reduction for bacteria and a 3-log reduction for fungi, is
less stringent than the requirements of the Quantitative Suspension
Tests (i.e., a 5-log reduction for bacteria and 4-log reduction for
fungi). This is to reflect the normally expected reduction in the
susceptibility of organisms to disinfectants as a consequence of
being dried onto the surface.

It has been recognized that the conditions used to dry the

organisms onto the surface of the stainless-steel coupons (37°C for
one hour) can result in a loss of viability with Gram negative and
yeast cultures. This can be significant and can result in a sufficient
loss of viability that may render it difficult to satisfy the acceptance
criteria (4-log reduction). In an attempt to improve viability, the latest
edition of the EN 13697 standard uses a higher inoculum level for
P. aeruginosa and C. albicans with BSA as the interfering substance.

EN 16615: 2015 – Phase 2/Step 2 – Quantitative

Non-Porous Surface Test with Mechanical Action
This test is used to establish whether a chemical disinfectant for use on
surfaces administered with wipes has a bactericidal and yeasticidal
activity. The test is intended for assessment of disinfectants used
in medical areas. The test closely simulates practical conditions of
application such as contact time (minimum one minute, maximum
five or 60 minutes), temperature (4°C to 30°C) and interfering
substances (clean conditions 0.3 g/L BSA; dirty conditions 3.0 g/L
 Validation of Disinfectants 407

BSA + 3.0 ml/L sheep erythrocytes). The conditions are intended

to cover general purposes in the medical area (hence the inclusion
of erythrocytes as an interfering substance). However, if for some
applications the recommendations of use of a product differ
additional test conditions may be used.

A large test surface (50cm × 20cm) is marked with four squares

of 5 × 5 cm, the “test fields”, in a row. Test field 1 is inoculated with
a test suspension of bacteria or yeasts in a solution of interfering
substances and allowed to dry. A wipe soaked with the test
disinfectant is wiped across the four marked test fields and, after
the contact time, neutralization and recovery of the test organisms is
performed, and the number of survivors is calculated. Bactericidal
activity is evaluated using: S. aureus, E. hirae and P. aeruginosa (5-
log reduction in test field 1, and mean CFU on test fields 2 to 4 of
<50 CFU); yeasticidal activity is evaluated using C. albicans (4-log
reduction in test field 1, and mean CFU on test fields 2 to 4 of <50
CFU). The increased acceptance criteria compared to EN 13697
+A1 2019 reflects the expected additional bioburden reduction as a
consequence of the mechanical action of wiping.

AOAC TEST METHODS

In the US, the official disinfectant testing methods are published
by AOAC International and include the Use Dilution Methods,
Sporicidal Activity Method, and Germicidal Spray Method. Data from
these tests are used in the Environmental Protection Agency (EPA)
registration process of products for sale in the US, as per the Federal
Insecticide, Fungicide, and Rodenticide Act. These methods are not,
however, recommended for validation of cleanroom disinfectants.
Only disinfectants capable of a high degree of biocidal activity in a
relatively short time are likely to pass these tests. The Use Dilution
Methods (955.14 Salmonella enterica; 955.15 Staphylococcus aureus;
964.02 Pseudomonas aeruginosa) and Sporicidal Activity Method
(966.04 Bacillus subtilis, Clostridium sporogenes) are semi-quantitative,
involve complete immersion of the carriers in the disinfectant and
lack any mechanical action, so do not reflect how the disinfectant
408 Cleanroom Contamination Prevention and Control

would be used in a cleanroom setting. Briefly, inoculated carriers

are deposited into the disinfectant. Following the contact time, the
carriers are deposited into a liquid neutralizing subculture medium
and are incubated. Each of the 60 tubes is visually inspected for the
presence or absence of growth, with specific performance standards
built for each test organism (e.g., 59 out of 60 negative for growth).
The methods therefore do not reflect how the disinfectant would
be used in a cleanroom setting and do not provide log reduction
calculations. The Germicidal Spray Method (961.02 Salmonella
enterica, Staphylococcus aureus, Pseudomonas aeruginosa, Trichophyton
mentagrophytes) can incorporate mechanical action by spraying
inoculated surfaces, and is not an immersion test, but only provides
qualitative results similar to the Use Dilution Method. The main
disadvantage of all of these methods is that their performance
requires a very high degree of skill, as the analyst must manipulate
60 carriers and move them between the solutions at high speed
without touching the sides of the containers or contaminating them.
As a consequence, there can be a high degree of variability in test
results. Their lack of log reduction calculations also makes them a
poor choice for cleanroom validation testing.

ASTM E2111
This is a fully quantitative surface test method, which uses the flat
inside bottom of glass vials as the test surface. Unlike European
surface test methods, this method also includes determination of
sporicidal activity.

A known amount of inoculum is dried onto the test surface

before being exposed to a known amount of test disinfectant for a
set contact time. At the end of the contact time, the disinfectant is
neutralized, and the organisms are physically removed from the
surface using a stirrer bar and enumerated by membrane filtration.
The test may be performed in the presence/absence of interfering
substance and also takes into account the test temperature.
 Validation of Disinfectants 409

The test is designed to have survivors and provides guidance on

calculation of results but does not specify acceptance criteria and is
not currently recognized by the US EPA in support of disinfectant
registration.

However, this test method is fully quantitative and, unlike the

AOAC methods described above, avoids any loss of viable organisms
through wash off, making it possible to produce statistically valid
data using many fewer test and control carriers than the AOAC
methods based on most probable numbers. The main disadvantage
of this test is that the test surface is the inside of the test container,
therefore it is not suitable for demonstrating efficacy on a variety of
cleanroom surfaces.

ASTM E2197 Standard Quantitative Disk Carrier

Test Method for Determining Bactericidal, Virucidal,
Fungicidal, Mycobactericidal, and Sporicidal Activities
of Chemicals
This is a quantitative surface test method, which uses 1 cm diameter
stainless steel carriers as the test surface. Unlike the European
surface test methods, this method also includes determination of
sporicidal activity.

A known amount of inoculum is dried onto the test surface

before being exposed to a known amount of test disinfectant for a
set contact time. At the end of the contact time, the disinfectant is
neutralized, and the organisms are removed from the surface by
vortexing and enumerated by membrane filtration. The test may be
performed in the presence/absence of interfering substance and also
takes into account the test temperature.

The test is designed to have survivors and provides guidance

on calculation of results but does not specify acceptance criteria. The
design of this test eliminates any loss of viable organisms through
410 Cleanroom Contamination Prevention and Control

wash off, thus making it possible to produce statistically valid data

using many fewer test carriers than needed for the AOAC methods
based on simple most probably number estimates.

At the time of writing the EPA has proposed, but not yet fully
adopted, an alternative method as the primary means to evaluate
limited, broad-spectrum, and hospital disinfectants in support of
public health claims. This method is based on E2197 and OECD
Guidance Document on Quantitative Methods for Evaluating
the Activity of Microbicides used on Hard Non-Porous Surfaces.
The alternative method has been adopted for two specific claims
regarding however, disinfection of C. difficile spores and Candida
auris. The method requires at least 6-log reduction of C. difficile
spores, and at least 5-log reduction for Candida auris. However, the
performance standard for general disinfection claims has not yet
been finalized.

SUMMARY
This chapter has considered the various standards that are available
to guide the microbiologist through the validation process and has
drawn out the difference between them. The particular methods
chosen for the validation study depend on the needs of the facility
and the intended use of the disinfectant. It is important that the lead
microbiologist in each organization plays an active part in selecting
the appropriate standard.

What these standards have in common, and in keeping with

the objective of validation, is ensuring that the in-use disinfectant(s)
are the most effective for supporting a facility contamination
control strategy. With respect to this, the surface tests are the most
meaningful tests to conduct. This must be followed by a supporting
field trial.
 Validation of Disinfectants 411

REFERENCES
European disinfectant standards
EN 12353:2020: Chemical disinfectants and antiseptics – Preservation
of test organisms used for the determination of bactericidal,
mycobactericidal, sporicidal and fungicidal activity.

EN 1650:2019: Chemical disinfectants and antiseptics – Quantitative

suspension test for the evaluation of fungicidal or yeasticidal
activity of chemical disinfectants and antiseptics used in food,
industrial, domestic and institutional areas – Test method and
requirements (phase 2, step 1).

EN 1276:2019: Chemical disinfectants and antiseptics – Quantitative

suspension test for the evaluation of bactericidal activity of
chemical disinfectants and antiseptics used in food, industrial,
domestic and institutional areas – Test method and requirements
(phase 2, step 1).

EN 1656:2019: Chemical disinfectants and antiseptics – Quantitative

suspension test for the evaluation of bactericidal activity of
chemical disinfectants and antiseptics used in the veterinary
area – Test method and requirements (phase 2, step 1).

EN 13704:2018: Chemical disinfectants – Quantitative suspension test

for the evaluation of sporicidal activity of chemical disinfectants
used in food, industrial, domestic and institutional areas – Test
method and requirements (phase 2, step 1).

EN 14885:2018: Chemical disinfectants and antiseptics – Application

of European Standards for chemical disinfectants and antiseptics.

EN 13623:2017: Chemical disinfectants and antiseptics – Quantitative

suspension test for the evaluation of bactericidal activity against
Legionella of chemical disinfectants for aqueous systems – Test
method and requirements (phase 2, step 1).
412 Cleanroom Contamination Prevention and Control

EN 12791:2016 +A1 2017: Chemical disinfectants and antiseptics

– Surgical hand disinfection – Test method and requirement
(phase 2, step 2).

EN 1657:2016: Chemical disinfectants and antiseptics – Quantitative

suspension test for the evaluation of fungicidal or yeasticidal
activity of chemical disinfectants and antiseptics used in the
veterinary area – Test method and requirements (phase 2, step 1).

EN 13697: 2015+A1:2019: Chemical disinfectants and antiseptics

– Quantitative non-porous surface test for the evaluation of
bactericidal and/or fungicidal activity of chemical disinfectants
used in food, industrial, domestic and institutional areas – Test
method and requirements without mechanical action (phase 2,
step 2).

EN 16615:2015: Chemical disinfectants and antiseptics. Quantitative

test method for the evaluation of bactericidal and yeasticidal
activity on non-porous surfaces with mechanical action
employing wipes in the medical area (4-field test). Test method
and requirements (phase 2, step 2).

EN 14675:2015: Chemical disinfectants and antiseptics – Quantitative

suspension test for the evaluation of virucidal activity of chemical
disinfectants and antiseptics used in the veterinary area – Test
method and requirements (phase 2, step 1).

EN 14476:2013+ A2 2019: Chemical disinfectants and antiseptics –

Virucidal quantitative suspension test for chemical disinfectants
and antiseptics used in human medicine – Test method and
requirements (phase 2, step 1).

EN 13624:2013: Chemical disinfectants and antiseptics – Quantitative

suspension test for the evaluation of fungicidal activity of
chemical disinfectants for instruments used in the medical area
– Test method and requirements (phase 2, step 1).
 Validation of Disinfectants 413

EN 1499:2013: Chemical disinfectants and antiseptics – Hygienic

handwash – Test method and requirements (phase 2, step 2.)

EN 1500:2013: Chemical disinfectants and antiseptics – Hygienic

handrub – Test method and requirements (phase 2, step 2).

EN 13727:2012 + A2 2015: Chemical disinfectants and antiseptics –

Quantitative suspension test for the evaluation of bactericidal
activity of chemical disinfectants for instruments used in the
medical area – Test method and requirements (phase 2, step 1).

EN 14349:2012: Chemical disinfectants and antiseptics – Quantitative

surface test for the evaluation of bactericidal activity of chemical
disinfectants and antiseptics used in veterinary area on non-
porous surfaces without mechanical action – Test method and
requirements (phase 2, step 2).

EN 14204:2012: Chemical disinfectants and antiseptics – Quantitative

suspension test for the evaluation of mycobactericidal activity
of chemical disinfectants and antiseptics used in the veterinary
area – Test method and requirements (phase 2, step 1).

EN 14563:2008: Chemical disinfectants and antiseptics – Quantit-

ative carrier test for the evaluation of mycobactericidal or
tuberculocidal activity of chemical disinfectants used for
instruments in the medical area – Test method and requirements
(phase 2, step 2).

EN 14561:2006: Chemical disinfectants and antiseptics – Quantitative

carrier test for the evaluation of bactericidal activity for
instruments used in the medical area – Test method and
requirements (phase 2, step 2).

EN 14562:2006: Chemical disinfectants and antiseptics – Quantitative

carrier test for the evaluation of fungicidal or yeasticidal activity
for instruments used in the medical area – Test method and
requirements (phase 2, step 2).
414 Cleanroom Contamination Prevention and Control

EN 14348:2005: Chemical disinfectants and antiseptics – Quantitative

suspension test for the evaluation of mycobactericidal activity of
chemical disinfectants in the medical area including instrument
disinfectants – Test methods and requirements (phase 2, step 1).

EN 14347:2005: Chemical disinfectants and antiseptics – Basic

sporicidal activity – Test method and requirements (phase 1).

EN 1275:2005: Chemical disinfectants and antiseptics – Quantitative

suspension test for the evaluation of basic fungicidal or basic
yeasticidal activity of chemical disinfectants and antiseptics –
Test method and requirements (phase 1).

EN 1040:2005: Chemical disinfectants and antiseptics – Quantitative

suspension test for the evaluation of basic bactericidal activity
of chemical disinfectants and antiseptics – Test method and
requirements (phase 1).

EN13610:2002: Chemical disinfectants – Quantitative suspension test

for the evaluation of virucidal activity against bacteriophages of
chemical disinfectants used in food and industrial areas – Test
method and requirements (phase 2, step 1).

US Disinfectant Standards
In the US, publications or regulatory guidance/standards such as
Chapter <1072>, “Disinfectants and Antiseptics” in the United States
Pharmacopeia address the issue of disinfectant testing but do not
state which method to use. For methods, those issued by the AOAC
tend to be followed, although this is not mandatory. Other sources
include the US EPA and the ASTM (American Society for Testing
and Materials).
 Validation of Disinfectants 415

Types of disinfection methods include the following:

AOAC Use-dilution method.

AOAC Hard surface carrier test method.

AOAC Germicidal Spray Products.

ASTM Standards E2197, 2017 Standard Quantitative Disk Carrier

Test Method for Determining the Bactericidal, Virucidal,
Fungicidal, Mycobactericidal and Sporicidal Activities of Liquid
Chemical Germicides.

ASTM Standards E2111, 2012 (2018) Standard Quantitative Carrier

Test Method to Evaluate the Bactericidal, Fungicidal, Mycobact-
ericidal, and Sporicidal Potencies of Liquid Chemical Microbicides.

ASTM Standards E2315 Standard Guide for Assessment of

Antimicrobial Activity Using a Time-Kill Procedure.

OECD Guidance Document on Quantitative Methods for Evaluating

the Activity of Microbicides Used on Hard Non-Porous Surfaces
(June 21, 2013).

This chapter was originally written for Pharmig and is reprinted with permission.

ABOUT THE AUTHORS

Tim Sandle is Head of Microbiology, Risk Management and Sterility
Assurance at BPL. He is also a visiting lecturer at the University
of Manchester and University College London. A member of the
Pharmig committee since 2002, Tim has run a Pharmaceutical
Microbiology website since 2010. Tim has written or edited 30 books,
165 peer reviewed papers and 450 technical articles pertaining to
microbiology, healthcare and pharmaceutical processing.
416 Cleanroom Contamination Prevention and Control

Rachel Kirkham With more than 30 years in the contamination

control industry, Rachel has held a variety of roles, including sales
leadership and technical consultancy. She has worked across the life
sciences sector, including for The Body Shop International, Tristel,
Diversey and she currently holds the position of Director of Global
Technical Consulting for Contamination Control and Validation at
Ecolab Life Sciences. In this role, Rachel leads a technical team that
provides expert technical support to pharmaceutical manufacturers,
including technical consulting on cleaning and disinfection and
guidance for best practice implementations, disinfection validation
studies and contamination control projects. Rachel is a regular
technical speaker on cleanroom control and disinfectant efficacy
validation. She has been a Pharmig committee member for 28 years.
As part of the Pharmig organisation, she has co-developed many of
the industry disinfectant training materials and tools.

Laura Guardi has over 20 years’ experience in the pharmaceutical

industry, and is currently an Associate QA Director in the Global
Quality Audit team at AstraZeneca. Laura has been a member of the
Pharmig committee since 2015.

Kim Morwood In a career spanning over 35 years in the industry,

Kim has previously worked for GSK and Sanofi in a variety of roles,
before becoming the Technical Director of MGS Laboratories Ltd in
2006. MGS is a contract testing laboratory which specializes in the
antimicrobial evaluation of disinfectant products and in particular the
current European CEN test methods. Kim also serves on the committee
of the Pharmaceutical Microbiology Interest Group (Pharmig).
 14

CASE STUDIES FOR IMPROVING

A RISK-BASED CLEANING AND
DISINFECTION PROGRAM

Jim Polarine and Beth Kroeger

STERIS Corporation
Location
USA

There have been several times in our careers, during site visits, site
evaluations, and site audits, where we have observed opportunities
for significant improvement in cleaning and disinfection programs.
These observations cover the spectrum from problems with
disinfectant preparation and application, to facility design and
materials degradation. This book chapter covers several case studies
developed from our observations over the past 20 years.

MAKING THE DILUTION

A common issue observed at many facilities is the ability to dilute
the disinfectant or sporicide correctly when making the use dilution.
There have been many instances where manual mixing, or the “glug-
glug” method, has been observed. This outdated method is not only
costly and inaccurate but can lead to spilling and other safety issues.
The best way to dilute the product correctly is by measuring the

417
418 Cleanroom Contamination Prevention and Control

disinfectant or sporicide with either a graduated cylinder or by use of a

pre-measured unit dose pack, which is then added to a pre-measured
quantity of water in a bucket or carboy. It is best to use the highest
grade of water that you have available in the area when making up
the use-dilution, and many times that is Water for Injection (WFI)
or United States Pharmacopeia (USP)/European Pharmacopoeia (EP)
grade water. The better the quality of water, the lower the risk for
spreading any viable or non-viable contamination in the cleanrooms
from the water itself. Additionally, some disinfectant technologies
are designed to work better in higher purity water.

USE OF THE DISINFECTANT USE-DILUTION

IN CLEANROOM BUCKET ASSEMBLIES
There are numerous cleanroom application equipment and bucket
assemblies to choose from, with single-use mop systems using pre-
diluted mop heads becoming more prevalent. However, standard
one, two-, and three-bucket assemblies are what the majority of users
have in place. Two-bucket mop assemblies consist of disinfectant in
one or both buckets. The bucket in front contains disinfectant use-
dilution, which is the solution applied directly onto surfaces and
the bucket under the wringer is the rinse and waste bucket. Three-
bucket mop assemblies consist of disinfectant in the front bucket or
front two buckets with the middle bucket containing rinse solution,
and the bucket under the wringer is used for catching wrung out
rinse waste solution (Figure 1). In both scenarios, the disinfectant
may be used in the front bucket and purified water (PW) or WFI may
be used in the rinse buckets. However, it is a best practice to use the
disinfectant solution in all buckets, except for the bucket under the
wringer in three-bucket assemblies, rather than in the application
bucket alone. Using WFI or PW in the rinse bucket could potentially
cause issues such as dilution of the disinfectant in the disinfectant
application bucket, possibly leading to efficacy concerns, or microbial
contamination issues since there is no disinfectant present to keep
contaminants in check. Using disinfectant in all but the waste bucket
avoids the problem of the operators inadvertently mopping with the
incorrect bucket.
 Case Studies for Improving a Risk-Based Program 419
Figure 1 Cleanroom triple-bucket application

APPLICATION METHOD
Another common detail that often gets overlooked is the correct
method of application in the cleanroom. When applying the
disinfectant use-dilution to the walls, floors, or ceiling in a cleanroom
with a flat head mop, the correct method of application is to go from
the most critical zones to the least critical. The application technique
should be mopping with unidirectional overlapping strokes by 10–
20%. You should never begin mopping in the gowning room and move
into the aseptic area as this would lead to spreading contamination
throughout the controlled areas. Utilizing clean equipment that does
not have rusting or pitting is also a critical element to an effective
program. The Food and Drug Administration (FDA) has recently
cited a cleanroom operation for using cleaning equipment that was
rusted and pitted (FDA, 2013). When using a string mop on the
floors mopping in a figure-eight pattern doing overlapping strokes
is very effective. Also, mopping should be done in a way that avoids
having the operator walk on wet floors. One of the most common
issues is poor mopping technique where the operator mops like they
would for home application rather than using proper technique.
This method supplies a visually clean surface, but simply spreads
the contamination around.
420 Cleanroom Contamination Prevention and Control

Sporicidal agent use is critical to an environmental control

program. Certain products used in this application may have either
an odor some facility cleaning personnel find objectionable or may
cause substrate damage with frequent use. The key to minimizing
either of these potential issues is to ensure that the product is used
correctly. In particular, ensure that the products are used at the
proper concentration, frequency, and with necessary strategies
to minimize personnel discomfort and substrate damage. The
volatility of these products in the environment may be controlled by
several factors such as adequate air changes per hour, temperature,
humidity, the application method, and the activity in the area.
Cleanroom areas may have approximately 20–50 air changes/hour.
Typically, maintaining 30 air changes/hour or greater is adequate
to reduce the build-up of volatile components of certain sporicidal
agents such as acetic acid/peracetic acid blends to acceptable levels
and reduces the need for the cleaning personnel to don respirators
or powered air-purifying respirators (PAPR). Temperature and
humidity are typically 16°C–24°C and 30–60% relative humidity.
Operation outside of these ranges may impact the evaporation
rate. Lower temperatures and higher humidity may reduce the
evaporation rate/drying time, potentially increasing the levels in the
environment. This is mitigated by maintaining the environment at
18°C–20°C and humidity less than 45% relative humidity.

Additionally, the application method and activity in the area

may contribute to higher than expected levels of volatile substances
in the environment. Mop systems where each mop head has a set
volume, such as pre-saturated mop heads may deposit an excessive
amount of product onto the surface. The recommended amount of
product per surface area is intended to ensure a clean solution is
available for the entire surface area to be covered. This amount does
not necessarily represent the amount of product required to cover a
surface such that it remains wet for a specified contact time. Using a
mop system with a wringer to remove the excess solution from the
mop head reduces excess amounts of disinfectant onto the surfaces,
which may minimize irritation from volatile components.
 Case Studies for Improving a Risk-Based Program 421

Use the correct equipment

Sites should maintain control of cleaning equipment by establishing
a review or approval team which evaluates and authorizes the use
of all materials and equipment used in the cleaning process. Review
and approval through this team, or material review board, should
include provisions for change control. Materials used in the cleaning
process should be evaluated for fit and purpose, ergonomics, and
compatibility with the cleaning agents. In one instance, a facility was
using cellulose sponges, containing an unidentified antimicrobial
technology intended to keep the sponges fresh for multiple uses.
While there is nothing inherently wrong with this technology, the
sponges are household commodities, not designed for cleanroom
applications. Household items are not always designed to be low-
particulating or to be free from other undesirable substances, such as
animal proteins. Further, these materials may introduce additional
chemical residues which could be deposited on product contact
surfaces during cleaning, or which could interfere with the products
being used for cleaning and disinfection rendering them less effective.
Examples of cleanroom tools observed in these environments include
brushes comprised of horse-hair and lambswool wipers. Both of these
items could leave behind animal-derived proteins, contaminating
a non-animal derived (non-ADR) environment. Preferably, select
industrial equipment designed for use in cleanrooms. These items
are designed to withstand exposure to more aggressive conditions,
such as chemical exposure and frequency of use, and generally hold
up better than household or janitorial applications. Mops should be
hung, so that the mop heads dry completely; alternatively select a
disposable mop head. Handles and mop buckets should be cleaned
after use or disposable liners in mop buckets should be used. Mops
should never be left in buckets with residual liquid at the bottom, nor
stored in this condition until the next use. Dirty solution left in a bucket
increases the occurrence of environmental excursions as it provides
an environment for organisms to proliferate, especially biofilm.
Cleaning equipment should be stored like any other equipment in
a critical environment: clean, dry, and covered or protected from the
surrounding environment. It is a best practice to use some type of
labeling or tracking system for traceability and to define a period of
422 Cleanroom Contamination Prevention and Control

use. Typically, application equipment such as brushes or mop heads

are used for a period up to one month. The fitness for reuse of this
equipment should be evaluated through visual inspection prior to
use. If particulate, dirt, or damage are noted, the item may not be
appropriate for further use.

Figures 2 and 3 Dirty mop bucket with mop stored in solution

CHANGING OUT THE USE-DILUTION

The second most common issue observed when cleaning and
disinfecting a cleanroom is ineffective use-dilution bucket change-
out procedures. The solution is meant to cover a specified surface
area, not to be used until depleted. The recommended bucket
change-out frequency is based on area covered per solution volume.
As a best practice, typically application area should be limited to
1,000 ft2 (93 m2) for a two- or three-bucket cleaning routine with use-
dilution or sporicide in each bucket for ISO-7 and ISO-8 cleanrooms.
The coverage area is every 600 ft2 for a two- or three-bucket cleaning
routine with use-dilution or sporicide in each bucket for ISO-5 and
ISO-6 cleanrooms (Dixon, 2009; IEST, 2019). The most conservative
approach is to change out the entire assembly (buckets, cart, mop,
mop head, and solution) or, at a minimum, empty the spent solution,
sanitize the buckets, or use bucket liners, and use a new mop head.
There have been several observations where the solution has become
soiled with floating debris, which is a sign the buckets need to be
changed out to avoid more contamination. In fact, in most procedures
reviewed, there is no indication of how long a use-dilution may be
 Case Studies for Improving a Risk-Based Program 423

used or when to discard the dirty solution, which may contribute

to residue and/or contamination issues. It is highly recommended
to incorporate surface area and disinfectant solution expectations
in the site’s cleaning Standard Operating Procedures (SOPs) or
Work Instructions (WI). Large rooms and long corridors may have
to be divided into grids with new bucket assemblies and fresh use-
dilutions prepared to cover the entire surface area. Areas should be
cleaned in a grid pattern and contact time measured for each area. If
any area of the floor is inaccessible, the disinfectant solution should
be sprayed under the equipment or area. Areas should remain wet for
the contact time given on the label or determined by examining the
efficacy against the in-house isolates via a disinfectant efficacy study.

CLEANING VERSUS DISINFECTION

During routine cleaning and disinfection procedures in the
cleanroom, it is typically not required to have a separate cleaning step
before disinfection. Disinfectants are formulated with surfactants
to enhance their cleaning ability and handle moderate soil loads in
cleanroom operations. Industrial disinfectants are intended to meet
their efficacy claims in the presence of a 5% organic soil (serum)
load, meaning they are meant to work in the presence of an organic
load, such as incidental spills, after equipment maintenance, and in
high traffic or transition areas. The exception would be after a worst-
case event such as a flood in the cleanroom (e.g., a pipe breaking)
or post construction. After these events, it may be necessary to use
a neutral or acidic cleaner for debris and soil removal, followed by
disinfectant application and a rinse step.

ROTATION
There have been numerous articles on rotation since about 1999. The
key to an effective cleaning and disinfection program is to have at least
one robust and effective disinfectant, such as a phenolic or quaternary
ammonium disinfectant, and one sporicide. The disinfectant
should be used on a routine basis for controlling bioburden on the
environmental surfaces in the cleanroom, and the sporicide should
424 Cleanroom Contamination Prevention and Control

be used less frequently based on environmental monitoring data

and the frequency of hits of fungal and bacterial spores (PDA, 2019;
USP, 2019). Therefore, sporicides may be used once a week, bi-
weekly, monthly, or quarterly, but the frequency of use should be
data-driven. The cleaning and disinfection program effectiveness is
assessed by the environmental monitoring data and the frequency of
excursions in the cleanroom.

Complicated procedures for cleaning and disinfection

We often review cleaning SOPs in order to provide input on industry
best practices. In many cases, there are several SOPs providing partial
or incomplete cleaning procedures and often they cross reference
each other with incorrect/inconsistent instructions. The SOPs may
raise confusion by allowing for the use of multiple disinfectants
and various dilutions of disinfectants and choices of disinfectant/
sporicidal agents that may be used, with very little clarification to
guide the user. It may be extremely difficult to discern the require­
ments for any area, which could lead to issues with SOP compliance.
To further exacerbate the issue with maintaining the cleanroom,
often there are no dedicated personnel to perform facility cleaning.
Facility cleaning routinely relies on manufacturing operators,
janitorial staff, or third-party contractors. It is a best practice to
have a dedicated team to perform facility cleaning/disinfection and
eliminate the use of a routine janitorial company to perform high-
level cleaning and disinfection. The personnel performing the facility
cleaning and disinfection must have specific training in cleanroom
access and behaviors, microbial control, sterility assurance, personnel
flow through the facility, and an understanding of disinfectant
efficacy, use of sterilants, safe handling, and application techniques.

Cleaning and disinfection procedures should be written to

ensure that operators can easily follow the procedure to avoid
deviations and ensure consistency. A best practice is to form a team
of subject matter experts (SME) from each area, so any concerns are
incorporated into the procedures. There should not be multiple ways
to clean areas of similar classifications unless they pose a higher risk
 Case Studies for Improving a Risk-Based Program 425

as determined by a risk assessment. Even then, it should be easy

to identify areas of higher risk such as Grade D areas adjacent to
uncontrolled areas or Grade C areas adjacent to Grade A/B areas.
Avoid using multiple disinfectants, dilutions, sanitizers, or allowing
operators to choose from multiple products. It is recommended
to use only one or two disinfectants, depending on the regulatory
expectations for the site, and to define the combination of products
to use and when. When choosing products for the environmental
control program, review the products claims versus in-house isolates
and ensure the product is being used correctly per those claims or
per disinfectant efficacy testing.

Rotating products should make sense and should be scientifically

justified. While it may not be necessary to rotate two disinfectants, if
the decision is made to do so, product selection and frequency of
rotation should be data-based. We have seen complicated procedures
where two disinfectants and a sporicidal agent were rotated with
one disinfectant being used each month (e.g., one full month of daily
application of a sporicidal agent followed by two months of daily
application of disinfectant with no sporicidal use during this interval).
Environmental monitoring (EM) trends indicated the site started
having issues six weeks after use of the sporicidal agent, i.e., during
the disinfectant application period where no sporicide was being
used; as a consequence, they considered increasing the frequency
of the sporicidal application. After reviewing the EM data and their
rotation program, it was determined they could reduce using the
sporicidal agent to one application per month with the disinfectant
being used daily in between as a more appropriate procedure.

CONTROLLING RESIDUES
Environmental control of a cleanroom is maintained by the frequent
application of disinfectants and sporicidal agents. However, routine
use of these products may leave behind residues. It is recommended
to periodically remove these residues from cleanroom surfaces since
they can build up over time and possibly contribute to functional,
particle, efficacy, and aesthetic issues. Efficacy issues may occur due
426 Cleanroom Contamination Prevention and Control

to incompatible disinfectant use in a rotation program where one

disinfectant either dilutes or inactivates the other due to residues
remaining on the surface between applications. Other considerations
include questions about the impact of the residues of various
products on each other and on microbial growth promotion, e.g., does
disinfectant residue alone promote microbial contamination, and does
the residue interfere with sporicidal agents or other disinfectants?

Residue from next-generation phenolic disinfectants were

evaluated to assess any impact on the efficacy of a sporicidal agent
against Bacillus subtilis, ATCC# 19659. In this case, phenolic residues
were introduced into Spor-Klenz RTU, which was subsequently
evaluated in a time kill study to determine impact on performance
against the spore-former. The quantity of residue, i.e., solids (µg/cm2)
from dried disinfectant was determined in a separate study where
the amount of residue was calculated after several applications
onto a 116 cm2 stainless steel test coupon to mimic typical use in the
cleanroom. Additionally, the amount of Spor-Klenz RTU to cover
a surface in typical applications was evaluated to determine the
volume required as the diluent. It was determined that 120 mg of
Spor-Klenz RTU sterilant was required to cover a 116 cm2 coupon.
The ratios determined in this experiment were extrapolated to
determine the dilution of disinfectant residue to spike into a solution
of a sporicidal agent for the time-kill study.

An amount of 0.1 mL of the microorganism suspension was

added to 9.9 mL of the test solution (i.e., Spor-Klenz RTU sterilant
spiked with either a low pH or high pH phenolic disinfectant). A
buffer control was spiked using the same amount. Post inoculation,
a 0.1 mL sample was removed at 2.5, 5, 7.5, 10, and 20 minutes. The
samples were neutralized, plated, and incubated for 48–49 hours at
37±2°C. The viable organisms per mL of sample were determined by
standard aerobic plate count.
 Case Studies for Improving a Risk-Based Program 427
Table 1 Mean log reduction of B. subtilis using phenolic residues
and sporicidal agent solution

Minutes, post inoculation 2.5 5 7.5 10 20

Mean log reduction: sporicidal 0.7 3.85 >4.16 >4.16 >4.23
agent + water
Mean log reduction: sporicidal 3.82 >4.15 >4.16 >4.16 >4.23
agent + low pH phenolic
disinfectant
Mean log reduction: sporicidal 3.32 >4.15 >4.16 >4.16 >4.23
agent + high pH phenolic
disinfectant

Similarly, dried disinfectant residues were evaluated to determine

the impact on sporicidal agent antimicrobial efficacy since this would
be more representative of typical cleanroom practices. Samples were
prepared to represent 20 days of disinfectant application to a known
area and allowed to dry, then treated with a volume of sporicidal
agent sufficient to cover the known area or 8.6 µL/cm2. The inoculum
was prepared from a spore suspension of Bacillus subtilis, ATCC#
19659, taken from stock culture. The test spore suspension was
diluted with deionized (DI) water to approximately 3.0 × 108 CFU/
mL. The suspension was added to tubes containing test samples or
Butterfield’s buffer to give a 1% v/v mixture of microorganism in test
product or buffer. At various time points, samples were neutralized,
plated, and incubated for 48–50 hours at 37±2°C post inoculum. The
viable organisms per mL of sample were determined by standard
aerobic plate count and transformed to log 10 values for analysis.

Table 2 Mean log reduction of B. subtilis using dried

phenolic residues and sporicidal agent solution

Minutes, post inoculation 2.5 5 10

Mean log reduction: sporicidal agent + water 0.94 2.94 > 4.02
Mean log reduction: sporicidal agent + low pH 1.22 > 4.04 > 4.02
phenolic disinfectant
Mean log reduction: sporicidal agent + high pH 1.75 > 4.04 > 4.02
phenolic disinfectant

Both disinfectant conditions resulted in enhanced sporicidal efficacy.

428 Cleanroom Contamination Prevention and Control

Recently, some auditors and cleanroom experts have indicated

disinfectant residue in a cleanroom environment is a serious issue,
and some even state a visually clean surface (on cleanroom surfaces)
is mandatory. While disinfectant residue can pose aesthetic issues
and should be removed from surfaces where the corrosiveness may
cause surface damage or look discolored, the study above indicates
there is no issue of the disinfectant residue interfering with the
sporicidal efficacy of the sporicidal agent.

Residue build-up should be monitored and countermeasures,

such as rinsing, should be increased if visible residue on the surface
becomes a problem. A cleaner may be applied in a manner that
is not in alignment with the typical application of a disinfectant,
i.e., using unidirectional, overlapping strokes. The objective of the
cleaning step is to loosen soil on the surface, so mechanical action
is preferred using circular motion with approved cleaning utensils
that are compatible with the cleaner. Cleaned surfaces are then
rinsed using WFI. It is not required to rinse a high-level cleaner
disinfectant after every application. The residue build-up in a month
is minimal compared to the residue from the routine application of a
cleaner. A routinely scheduled WFI rinse in a disinfectant program
will help to limit or even prevent residue build-up, minimizing the
need for a remedial clean. The frequency for rinsing is determined
by the disinfectant application frequency; for example, residual
disinfectant is typically removed by rinsing with WFI after 30
applications and/or before the application of the sterilant.

CONCLUSION
Many factors go into a successful risk-based cleaning and
disinfection program. The disinfectant dilution step is a critical
element along with using an effective one, two-, or three-bucket
cleaning routine to minimize disinfectant performance problems
which may arise from over-loaded solution. The application method
and frequency of disinfectant solution change out in the cleanroom
also have a significant effect on the overall control of bioburden
in the environment. Residual build-up in cleanrooms needs to
 Case Studies for Improving a Risk-Based Program 429

be removed on a routine basis, and attention needs to be given to

aesthetics and safety (sticky, tacky, and slippery) when determining
a rinsing frequency. The rotation of one or two disinfectants and a
sporicidal agent are globally accepted (PDA, 2019). The sporicidal
agent needs to be applied on a routine basis and should be based
on the number of hits from both fungal and bacterial spores. The
overall effectiveness of the cleaning and disinfection program
always improves with annual training and well-written SOPs. These
case studies are intended to be lessons for cleanroom operators and
managers to improve the overall compliance and robustness of the
cleaning and disinfection program.

REFERENCES
Dixon, A. (2009) “Cleaning of Non-Product Contact Surfaces,” in Paul
L. Pluta (Ed.) Cleaning and Cleaning Validation for the Pharmaceutical
and Medical Device Industries. Vol. 1 Basics, Expectations, and
Principles. Parenteral Drug Association, Inc., Bethesda, MD, and
DHI Publishing, LLC, River Grove, IL., p. 266.

Public Health Service Food and Drug Administration (FDA) Seattle

District Pacific Region, Warning Letter, November 27, 2013.
Available at https://www.fdanews.com/ext/resources/files/12/12-10-
13-JubilantWL.pdf

Institute of Environmental Sciences and Technology (IEST) (2019)

IEST-RP-CC018.4 Cleanroom Housekeeping: Operating and
Monitoring Procedures.

PDA (2019) PDA Technical Report No. 70. Fundamentals of Cleaning

and Disinfection Programs for Aseptic Manufacturing Facilities.
Parenteral Drug Association, Inc., Bethesda, MD.

USP/NF (2019) General Information Chapter <1072> Disinfectants

and Antiseptics. In United States Pharmacopoeia USP 42;
United States Pharmacopeial Convention/National Formulary,
Rockville, MD.
430 Cleanroom Contamination Prevention and Control

ABOUT THE AUTHORS

Mr. Polarine is a senior technical service manager at STERIS
Corporation. He has been with STERIS Corporation for 20 years
and his current technical focus is microbial control in cleanrooms
and other critical environments. Mr. Polarine is a 2019 PDA Michael
S. Korczynski Award recipient. He has lectured in North America,
Europe, Middle East, Asia, and Latin America on issues related
to cleaning and disinfection and microbial control in cleanrooms.
Mr. Polarine is a frequent industry speaker and published several
PDA book chapters and articles related to cleaning and disinfection
and contamination control. He is active as co-chair on the PDA’s
microbial investigations task force. He was a co-author on PDA’s
Technical Report #70 on Cleaning and Disinfection. Mr. Polarine
teaches industry regulators as well as the pharmaceutical, biotech,
and medical device industries at the PDA and the University
of Tennessee. Mr. Polarine currently teaches the cleaning and
disinfection course as part of the PDA’s Aseptic Processing Course
and at the University of Tennessee’s Parenteral Medications Course.
He is current President for the PDA Missouri Valley and Technical
Coordinator for the IEST. Mr. Polarine graduated from the University
of Illinois with a Master of Arts in Biology. He previously worked
as a clinical research coordinator with the Department of Veterans
Affairs in St. Louis, MO and as a biology and microbiology instructor
at the University of Illinois. His main hobby is storm chasing and is
very active in tornado research and tornado safety.

Beth Kroeger is a Technical Services Manager for the Life Sciences

Division of STERIS Corporation. She currently provides global
technical support related to process research cleaners, cleaning
validation and critical environments and frequently speaks on these
topics for the Institute of Validation Technology (IVT), United States
Pharmacopeia (USP), International Society for Pharmaceutical
Engineering (ISPE), Parenteral Drug Association (PDA) and the
National Institute for Bioprocessing Research and Training. Beth has
over 25 year’s industry experience in Biopharmaceutical and Oral
Solid Dose manufacturing operations. Prior to her current position,
 Case Studies for Improving a Risk-Based Program 431

Beth was employed by Janssen Biotech where her areas of expertise

included large scale bioreactor systems, both continuous perfusion
and fed batch, and downstream operations including large scale
purification processing, viral removal, UF and final fill. Beth has held
positions as an Operations Manager, Senior Compliance Associate
and as the manufacturing lead on a cross-functional team responsible
for the scale-up and technical transfer of 4 biopharmaceuticals. In
her role in manufacturing and compliance, she conducted hundreds
of investigations relating to excursions, malfunctions, failures and
errors. She earned a B.S. in Biological Sciences from the University
of Missouri, St. Louis and most recently completed a Fermentation
Technology program at Massachusetts Institute of Technology.
 15

CLEANROOM CLEANING –
CHOOSING, USING, AND
HANDLING CLEANING MATERIAL

Matts Ramstorp
BioTekPro AB
Sweden

INTRODUCTION
Contamination control, and especially cleanroom technology, rely
on cleaning and disinfection. This has been known for a long time
and is to a certain degree also the reason why cleanrooms were
developed and used within the air and space industry in the middle
of last century.

Cleaning is a task performed in our homes, and is, due to this

fact, considered as an inherited operation that everyone shall and
can perform. However, cleaning including disinfecting a cleanroom,
is a vital, critical, and highly specialized operation, that cannot be
performed in the same way as domestic cleaning, and the choice,
and handling of cleaning materials becomes extremely important.

433
434 Cleanroom Contamination Prevention and Control

WHY IS CLEANING OF CLEANROOMS

AND CLEAN ZONES CRITICAL?
Cleanrooms are used in industrial applications and within certain
areas of the health care sector to create a clean enough surrounding
air, in order to minimize the possibility to contaminate:

• The product.
• The processes.
• The personnel.

The overall aim using cleanrooms is that product produced or

handled will have the correct and desired quality attributes, i.e.,
work according to specifications and even in some cases, like within
the pharmaceutical and the health care sector, to minimize the risk
for harming a patient. Cleanrooms are defined in ISO 14644 Part 1
(ISO, 2015) as:

“A room within which the number concentration of airborne particles

is controlled and classified, and which is designed, constructed and
operated in a manner to control the introduction, generation and
retention of particles inside the room.”

This document also states:

“Levels of other cleanliness attributes such as chemical, viable or

nanoscale concentrations in the air, and also surface cleanliness in
terms of particle, nanoscale, chemical and viable concentrations might
also be specified and controlled.”

“Other relevant physical parameters might also be controlled as

required, e.g., temperature, humidity, pressure, vibration and electro­
static.”

A cleanroom is only defined by the number concentration of particles

in the surrounding air and cannot be defined based on particles
 Choosing, Using, and Handling Cleaning Material 435

contaminating surfaces. Even if this is the case there are in many

cases an absolute need to control surface contaminations, i.e., there
is a need to create clean surfaces.

Everything that is undertaken in a cleanroom and (or) a clean

zone will create contaminants, especially in the form of particles.
Furthermore, the process or the handling might cause spills of critical
liquid chemicals that will contaminate floors, process equipment,
clean zone work chambers etc.

Everything taking place or performed in a cleanroom will

influence the cleanliness of surfaces in the room. Since cleaning is
performed to create clean enough surfaces, the practical cleaning
steps must create clean enough surfaces. However, the cleaning
process must be performed in such a way that contaminants
(particles) will not be dispersed into the surrounding air and further
transported to other surfaces or critical parts of the room.

EVERYTHING IN A CLEANROOM
IS INTERCONNECTED
Even though the major purpose of a cleanroom is to create a clean
enough surrounding air, every action taken place in the room will
have an effect on the cleanroom as a whole, especially all the different
surfaces in the room.

The circle schematically illustrated in Figure 1 shows the four

major sources of contaminant (Ramstorp, 2000). These sources are:

• The personnel, i.e., the persons present in the room.

• The surrounding air in the room.
• The surfaces in the room, and finally
• The process and/or the product.
436 Cleanroom Contamination Prevention and Control
Figure 1 The relationships between the four major sources
of contaminants in an enclosed space

Source: Author

The major aim in using a cleanroom is normally to create a

clean enough environment to protect the product, in the case of
pharmaceutical cleanrooms – to protect the patient. Looking at the
circle in this figure, all four sources of contaminants can generate,
disperse or carry particles that might come in contact with and
thereby contaminate the product and compromise patient safety.

The most critical contaminants are generated by personnel.

Personnel are also the source generating most particles by numbers.
Particles from humans include skin debris, that are considered dead
particles, i.e., these particles can not reproduce. However, the human
body is covered with microorganisms, many of which belong to the
human normal flora. The released skin debris are therefore carrying
microorganisms and must be considered as “living” particles.

These released skin debris will to a certain degree be dispersed

out into the surrounding air of a cleanroom, and thereby contaminate
the air. The surrounding air will be in contact with the process
 Choosing, Using, and Handling Cleaning Material 437

(product) and there is a risk that the process (product) will be

contaminated by microorganisms located on these debris.

Particles dispersed in the surrounding air will behave differently.

The larger particles will fall downwards due to gravity, and will
settle on floors, tabletops, external process surfaces, etc. Smaller
particles might, due to differences in static electricity, be attracted
to other smaller particles and form aggregates that also might be
subjected to gravitational forces. Small particles might, due to
electrostatic forces, be attracted to surfaces having opposite polarity
and settle on all types of surfaces including walls and ceilings.
Particles in the surrounding air are, due to these reasons, one of the
major possibilities for creating contaminated surfaces.

Cleaning of surfaces in a cleanroom is, due what has been stated

above, a must. However, cleaning of surfaces will in many cases
also be a risk of generating and dispersing particles out into the
surrounding air. When wiping or mopping a surface, particles are
released from a surface. Many of these released particles are captured
by the wipe or mop, alternatively, but some of the released particles
will be dispersed into the surrounding air, giving these particles the
possibility to come in contact with the process (product) and (or)
settle once again on other more or less critical surfaces.

The fact that particles are dispersed into the surrounding air
during a cleaning process is not always recognised as a problem,
unfortunately.

VISIBILITY IS A MAJOR PROBLEM

DURING CLEANING AND DISINFECTION
A healthy human eye, without magnifying aids and in normal
indoor lightning, is said to have the possibility to identify individual
particles with a size greater to 40 micrometres. In practice this
means that most harmful particles, i.e., microorganisms or micro­
organism carrying particles, are invisible to the human naked
eye. In addition, it is impossible to, just by looking at a surface,
438 Cleanroom Contamination Prevention and Control

determine if the surface is clean enough or not, and this is the reason
why it is extremely difficult to clean a surface that looks clean from
the start. You can draw a parallel – cleaning a visibly clean surface is
like painting a surface with an invisible paint.

Visibility limitations also occur when focusing on air move­

ments. In the same way as smaller particles, i.e., below the visibility
limitations of the human eye, will not be identified in the surround­
ing air, it is impossible to identify how the flow of air inside a
cleanroom moves.

CLEANING IS OFTEN THE MOST CONTAMINATING

PROCESS IN A CLEANROOM
Due to the facts described above, cleaning is a critical process in
more than one aspect:

• The surface must be rendered clean enough.

• The process of cleaning might contaminate the surrounding air,
and thereby re-contaminate surfaces in the cleanroom.

Both these two aspects must be taken into consideration when

discussing cleaning.

Figure 2 shows schematically how the particle concentration in

the surrounding air will change during a production process in a
cleanroom. The y-axis shows particle concentration measured with
an optical particle counter and the x-axis shows time during a fictive
production.

In the morning, the particle concentration will be extremely

low, corresponding to the at rest state of the cleanroom. At point
A the operators enter the cleanroom to set up the process and start
their work. The particle concentration will increase and reach a semi
steady state situation during the entire production time. At point B
production is ended and the cleanroom is to be cleaned. At point C
 Choosing, Using, and Handling Cleaning Material 439

cleaning starts, and as shown in the figure the particle concentration

is increasing. This increase is due to what was previously mentioned,
i.e., particles on surfaces are released and not fully collected by the
cleaning process or material. The released and not captured particles
will instead be dispersed into the surrounding air. The increase in
particle concentration might also be due to the physical movement
of the cleaning operators, as compared to the operators performing
production.

Figure 2 Change of particle concentration in air

Source: Author

HOW TO MINIMIZE DISPERSION OF

PARTICLES DURING CLEANING
From what has been stated up till now, it is possible to minimize the
particle concentration increase during cleaning by focusing on the
following:

• Choice of cleaning technique.

• Choice of cleaning material.
440 Cleanroom Contamination Prevention and Control

• Time allowed for cleaning.

• Cleaning operator training.
• Cleaning operator behavior.

A QUICK AND HELPFUL WAY TO

DETERMINE IF A CLEANING TECHNIQUE
IS SUITABLE FOR CLEANROOM USE
One way of determining if a technique is suitable for cleanroom
cleaning is to look at the technique and try to answer the following
questions:

• Does the technique detach particles from a surface?

• Is it possible to capture the released particle? And finally
• Is it possible to move the capture particle out of the cleanroom
or away from a more critical area to a less critical area?

If all these questions are answered with a yes, there is a possibility

the technique in question will be suitable for cleanroom cleaning,
and furthermore, to study it from a more practical point view.

Wiping and mopping are techniques that will fulfil the three
demands above, whereas, for example using pressurized air release
particles by blowing of a surface will only fulfil the first demand, to
a certain degree, and will due to natural reasons not fulfil the two
later ones.

CHOICE OF CLEANING MATERIAL

The major demand for materials to be used in cleanrooms follow the
same strict rule. Everything taken into a cleanroom should, from a
general point of view, be …
 Choosing, Using, and Handling Cleaning Material 441

• Clean enough.
• Not releasing particles or fibres.
• Packed in such a way to facilitate sanitation when bringing
material from a lower or non-classified area into the area in
which it is to be used.

All these three demands are dependent on one and the same basic
criterion – the cleanliness of the area in which the material is to be
used.

TIME ALLOWED FOR CLEANING

Cleaning a cleanroom can never be compared to domestic cleaning.
The cleanroom ISO standard ISO 14644 includes a document, Part 5
with the title: ISO 14644–5 “Cleanrooms and associated controlled
environments — Part 5: Operations” (ISO, 2004). Even though this
document is quite old, it contains some fundamental and extremely
important knowledge on cleaning, that fundamentally illustrates the
difference between domestic and cleanroom cleaning.

First, it helps to identify and classify surfaces depending on

their respective criticality, in defining the following:

• Critical surfaces.
• General cleanroom surfaces.
• Other surfaces.

Without going more deeply into these different surfaces, it is

possible to conclude that all surfaces are not considered equal from
a criticality perspective.

Second, this standard also specifies and defines three levels of

surface cleanliness in respect to the elimination of particles, and,
furthermore, particle sizes are stated for these three levels:
442 Cleanroom Contamination Prevention and Control

• Gross cleaning, which is specified as removal of particles with a

size >50 micrometre.

• Intermediate cleaning, removal of particles in the region between

10 and 50 micrometres.

• Precision cleaning, removal of particles with a size <10

micrometre.

The ISO cleanroom standard ISO 14664 is, from a general point of
view, based on particles, and microbiological contaminants are not
considered at all in this standard, However, it is possible to view the
above stated definition of surface cleanliness from a microbiological
point of view. Gross cleaning as well as intermediate cleaning are
both valid when working with microbiological contamination
control in cleanrooms, and under these circumstances the term
precision cleaning is normally translated as disinfection.

HOW TO CLEAN
The practical part of cleaning and disinfection varies from activity
to activity mostly due to the desire for cleanliness and need of the
cleanroom. Cleanrooms are generally more critical as compared to
airlocks leading to and from the cleanroom, but the cleanliness level
in each of these, cleanrooms as well as airlocks, varies.

The traditional way to clean involves the use of water, often

with the addition of various chemicals. The different chemicals
used can be in the form of one (clean) chemical alone, or a mixture
of different chemicals with different abilities to either free particles
from surfaces, or dissolve dry residuals from the surface. In general
all these chemicals or mixtures of chemicals must be validated
in order to not only determine that they are compatible with the
surface material to be cleaned, but also to determine that the wipe
and (or) mop chosen is compatible as well.
 Choosing, Using, and Handling Cleaning Material 443

CLEANING OPERATOR TRAINING

The European GMP consists of several volumes, and each of these
includes several annexes. One of the most discussed annexes at
present is the one that normally is called “Annex 1”. This annex
is from a more formal point of view. Annex 1 is found in Volume
4, with the full title: “EudraLex – The Rules Governing Medicinal
Products in the European Union”, Volume 4 “EU Guidelines to
Good Manufacturing Practice – Medicinal Products for Human and
Veterinary Use”, and Annex 1 –“Manufacture of Sterile Medicinal
Products” (EudraLex, 2008).

“Annex 1” is presently undergoing revision, and this process

has taken quite a long time. The latest version, version 12, was made
public in the beginning of 2020, and the final approved document
might, hopefully, be published in 2021. All draft versions have had
a focus on cleanroom operators, and to a certain degree on their
training and knowledge. Future demands include that operators,
both production operators as well as cleaning operators do not only
need a good enough “know-how”, but also a thorough “know-
why”. The more knowledge the better, to create not only skilled
operators but also operator motivation to perform correct and well.

In many cases cleaning operators are not given the correct or

needed knowledge in contamination control, cleanroom technology,
basic microbiology, aseptic production, etc. This is pinpointed out in
the “Annex 1” to come, as extremely important tools for the opera-
tors, independent on their respective function.

CLEANING OPERATOR BEHAVIOR

In the same way as the demands for material to be introduced into
cleanrooms must fulfil strict demands, operator behavior follows
approved rules and regulations. When it comes to behavior, process
operators as well as cleaning operators, are not allowed to move at a
444 Cleanroom Contamination Prevention and Control

rapid pace, all movements should be calm and controlled (Ramstorp,

2011). This is of great importance when discussing cleaning of
cleanrooms, since the process of cleaning, either wiping or mopping,
involves much more physical mobility as compared to maintaining
a cleanroom production process.

The time set for cleaning a cleanroom has a direct impact of

cleaning operator behavior – the less time set for cleaning, the faster
the pace of the cleaning operator, and as a consequence – the higher
risk for mistakes and, especially higher levels of particles dispersed
into the surrounding air of the cleanroom.

To summarize this first part of the chapter, cleaning a cleanroom

is in many cases considered the most contaminated activity in a
cleanroom. The increased particle concentrations shown during the
cleaning process in Figure 2 is critical to the cleanliness of not only
the cleanroom but also all the activities that are to be performed in
the cleanroom at a later stage, due to the possible sedimentation of
released particles.

One way of securing the cleanroom and thereby also the production
and the product from particles released during cleaning is to
allow the cleanroom to recover after cleaning. The clean-up of the
surrounding air in a cleanroom is accomplished by introducing
cleaner filtered ventilation air to the cleanrooms. This clean-up
period is in the US mainly based on air turnover rates, i.e., the
number of times per hour that the total volume of air in a cleanroom
is exchanged and replaced by cleaner ventilation air. In Europe, on
the other hand, air exchange rates were defined in older versions of
“Annex 1”, but these exchange rates have now been replaced by the
term “recovery time”. Recovery time can be described as the time
needed for a cleanroom with a high concentration of particles in the
surrounding air to, when the cleaning operators have left, Point D
in Figure 2, by the introduction of cleaner ventilation air, to reach its
“at rest” state.
 Choosing, Using, and Handling Cleaning Material 445

The difference between air exchange rate and recovery time

is distinct. Air exchange rate does not consider the distribution
efficiency of the introduced ventilation air in the room, and only
states how much air is introduced into the cleanroom. Recovery time,
on the other hand, covers both aspects, volume of air introduced as
well as the distribution efficiency of the introduced air.

The clean-up period after cleaning is only advantageous for the

cleanliness of the air in the cleanroom, due to the fact that quite a
lot of particles, and especially the larger, i.e., the heavier particles,
dispersed into the air during cleaning will settle onto, and thereby
contaminate different surfaces in the cleanroom.

CLEANING MATERIALS –
FUNDAMENTAL PROPERTIES
The two most universally used cleaning materials are wipers and
mops. Both can be used in a wet or a dry manner. They can be re-
useable or single use, supplied in a dry state to be soaked with water
and cleaning chemical at the site prior to use, or pre-saturated upon
delivery, and furthermore, they can be used in a clean or sterile state.
If adding these options to all the various demands stated earlier, it
is obvious that there are many different and, most of all, important.
decisions to make before even thinking of the actual cleaning process
itself.

Fundamental questions to state are:

• What cleanliness class or grade is to be cleaned?

• What is to be cleaned – cleanroom, air lock, pass through

hatches, etc.?

• What is to be cleaned – floor, walls, windows, tabletops,

cartwheels, process equipment, operator gloves, material to be
stepwise sanitized into the cleanroom, etc.?

• Is the cleaning performed in a dry or a wet state?

446 Cleanroom Contamination Prevention and Control

• Is there a minimum volume of chemicals needed per unit surface

area of cleaning or disinfection chemicals?

• What chemicals are to be used for cleaning?

• What chemicals are to be used for disinfection?

• Are there any special requirements for disposal of cleaning

materials after use? Etc?

CLEANROOM WIPES
Cleanroom wipes are used in order to remove contaminants, i.e.,
particles, liquid spills as well as contaminants. There are two types
of wipes available:

• Knitted or woven.
• Non-woven.

Knitted or woven wipes

Knitted or woven cleanroom wipes offers the cleanest level of wipes
and are most used in ISO 4 and ISO 5 cleanrooms. These wipes are
often manufactured from 100% polyester and are available in a
variety of edge finishes:

• Knife cut edge.

• Laser sealed edge.
• Ultrasonically sealed edge.

Laser or ultrasonically sealed edges apply for more critical wipes.

Important features of knitted or woven cleanroom wipes are:

• Strong, tear resistant and non-shedding.

 Choosing, Using, and Handling Cleaning Material 447

• Clean, low in particle release and can therefore be used in most

critical application and cleanrooms.
• Soft and non-abrasive.
• Sterilizable by gamma irradiation.
• Highly absorbing.
• Can have different edge finish.

Figure 3 Single-use wiper with cut edges

Source: Tiwiplusk

Figure 4 Reusable, washable knitted wipe with stitched edges

Source: Author
448 Cleanroom Contamination Prevention and Control

Non-woven cleanroom wipes

Non-woven cleanroom wipes are generally manufactured in two
ways:

• An extrusion process in which melt blown material forms a

sheet that is thermally bonded.

• A high-pressure water-based process creating hydroentangled

blended fibres.

Important features of non-woven cleanroom wipes are:

• Highly absorbing.
• Strong.
• Low linting.

Different types of blended non-woven wipes are available, for

example:

• Polypropylene/cellulose.
• Polyester/lyocell.
• Bonded polypropylene.
• Melt blown bonded polypropylene.
• Hydroentangled rayon/polyester.
• Spun laced polyester.

Polyester-cellulose wipes
These wipes are hydroentangled and offer a relatively good absorp­
tion capacity with low particle generation. Furthermore, they offer
high chemical resistance. Polyester-cellulose wipes are generally
used in ISO 1–9 cleanrooms (ISO 14644) and in Grade A–D cleanliness
classes for microbiologically controlled areas (Eudralex, 2008).
 Choosing, Using, and Handling Cleaning Material 449

Cotton wipes
Cotton wipes are relatively heat-resistant, electrostatically neutral,
absorbent, and largely resistant to acids and solvents.

CREATING A CLEANING PROGRAM

Cleaning cleanrooms and other types of controlled environment is
a delicate task (Ramstorp, 2011). The cleaning process is extremely
strict. First, the cleaning material has to be chosen correctly in
order to give the wanted cleanliness, the material must be capable
of releasing particles for the surface, have the capability to capture
the released particles as well as release a minimum of the captured
particles. Second, the cleaning material must be manufactured of a
material with minimum ability to release matter from the material
itself, due to chemical incompatibility and (or) physical when in
contact with the surface to be cleaned. Third, cleaning chemicals
must be chosen correctly, in respect of what type of contaminants
to be removed and the compatibility of the surface composition.
Finally, the cleaning operators work pattern – surfaces in cleanrooms
are normally not as dirty as surfaces at home. This means that the
cleaning operator must work with cleaning in a methodological
way, to clean the entire surface.

The overall aspects to consider when setting up a cleanroom

cleaning program includes:

• What is to be cleaned – cleanroom, personnel airlock, material

airlock, process equipment, pass-through hatches, etc?

• Classify all surfaces – critical surfaces, general cleanroom

surfaces, other surfaces.

• Choose the optimal cleaning method for the respective surfaces.

• Set frequencies of cleaning for the respective surfaces.

• Produce a cleaning timetable – what, when, how, by whom, etc?

450 Cleanroom Contamination Prevention and Control

• Who is to perform cleaning – production operators, cleaning

operators – internal or external personnel?

• Choose type of cleaning materials – single-use, re-useable or

combining both.

• Choose the desired cleaning materials – software as well as

hardware.

• Decide how and where to store cleaning materials – hardware.

• Decide how to document that cleaning has been performed.

• Decide how to control the outcome of the cleaning process.

Single use or re-useable cleaning materials

When setting up a cleanroom cleaning program from a cleaning
material point of view the choice between single-use and re-useable
is of fundamental importance. Many different aspects can and must
be considered, for example:

• Cleanliness demands – i.e., cleanroom class or grade.

• Type of production – non-hazard, biohazard, dangerous

chemicals, etc.

• Cleaning need – removal of particles, bio-contaminants, liquid

spills.

• Different cleaning chemicals needed – based on surface

composition, cleaning timetable.

• Type of surface to be cleaned – floors, walls, tabletops, process

equipment, etc?

• Storage capacity – new, unused versus storage of used prior to

recycling and waste disposal.
 Choosing, Using, and Handling Cleaning Material 451

• Time – handling of new as well as used reusable versus single

use.

• Chemical treatment of reusable versus untreated or pre-wetted

single use materials.

• Cost – including recycling and (or) disposal.

CLEANING OF CLEANROOMS FROM

A PRACTICAL APPROACH
As stated earlier, cleanrooms must be cleaned to an appropriate
level. Different cleanroom cleanliness classes or grades need differ­
ent surface cleanliness levels, which in turn means that all cleaning
processes are different. In practice, cleaning processes can have
different approaches. When using cleaning materials that are not
pre-treated, for example with cleaning chemicals, i.e., dry re-useable
and dry single use materials, the cleaning material must be treated
with the appropriate chemical prior to use. This pre-treatment is
normally and is in some cases a must, soaked in chemicals in the
cleanroom to be cleaned. If re-useable material is to be used it can also
be used several times, for example, when mopping a floor or wall,
but needs then to be rinsed and added to a new cleaning chemical in
order to continue the cleaning process. Alternative solutions can be
used in this context

• Pre-wetting of a pre-determined number of mops.

• The one-bucket system.
• The two-bucket system.
• The three-bucket system.
452 Cleanroom Contamination Prevention and Control

The use of a predetermined number of pre-wetted mops

Pre-wetting of a mop that is only used on a pre-determined floor area
is one alternative. The mops are normally prepared in the cleanroom
to be cleaned by preparing the cleaning solution in a bucket, adding
a certain volume of cleaning solutions to each mop, and allowing a
certain equilibration time for the solution to soak into the mop. Each
of these mops are then used for cleaning a pre-determined surface
area, after which a new pre-treated mop is used, and so on. The
advantage of this system is that a used mop is never re-used, and
there is no need to prepare excess volumes of cleaning chemicals.
Only a precise and predetermined volume of cleaning chemicals
needs to be prepared in order to soak the exact and predetermined
number of mops. A disadvantage of this methodology is that the
number of mops in use will be higher, although it is quite easy
to calculate the total number of mops needed by setting a certain
floor area per mop. The major advantage using the methodology
is that every mop that is prepared contains the same amount of
clean cleaning chemicals, and furthermore, the risk of moving
contaminants from one cleanroom floor are to others is a minimum.

The one-bucket system

The one-bucket system is based on preparing the cleaning chemical
in a bucket in the cleanroom to be cleaned. The mop to be used is
soaked in the cleaning chemical and used to mop a pre-determined
floor area. The same mop is then re-soaked in the bucket and
used to continue to clean the floor. The advantage of this cleaning
system is that one mop alone is used for the entire floor, but the
disadvantage is the cleaning chemical in the bucket is only clean
and fresh when the mop is soaked the first time. Eventual contamin­
ant released and captured by the mop will, to a certain degree be
added to the chemicals and distributed to the entire floor. This
cleaning system is not recommended in cleanrooms with higher
demands of surface cleanliness.
 Choosing, Using, and Handling Cleaning Material 453

The two-bucket system

The two-bucket system is fundamentally better as compared to the
one-bucket system, since the cleaning chemicals will be less con­
taminated during use (Sandle, 2010). The different buckets serve as:

• Bucket 1 – contains the cleaning solution to be used.

• Bucket 2 – serves as bucket for waste.

The process of using the two-bucket system shown in Figure 5

comprises of:

• Prepare the cleaning chemicals in Bucket 1.

• Soak the mop in the cleaning solution in Bucket 1.

• Wring the mop to remove excess cleaning solution into the waste
bucket – Bucket 2.

• Mop a pre-determined floor area with the mop.

• Soak the mop into the cleaning solution in Bucket 1.

• Wring the mop to remove excess cleaning solution into the waste
bucket – Bucket 2.

• Mop a pre-determined floor area with the mop.

• Repeat the above process.

The advantage of the two-bucket system is that one and the same
mop can be used for quite a large floor area. The disadvantage is that
the rinsing process only comprises of one step, which can lead to a
risk of spreading contaminants release from one floor area to another.
454 Cleanroom Contamination Prevention and Control
Figure 5 The two-bucket system process

Source: Author

The three-bucket system

The three-bucket system is a further development of the two-bucket
system, by adding a third step. The different buckets serve as:

• Bucket 1 – contains the cleaning solution to be used.

• Bucket 2 – serves as bucket for waste.
• Bucket 3 – contains clean water for rinsing purposes.

The process of using the three-bucket system shown in Figure 6

comprises of:

• Preparing the cleaning chemicals in the first bucket – Bucket 1.

• Soak the mop in the cleaning solution in Bucket 1.

• Wring the mop to remove excess cleaning solution into the waste
bucket – Bucket 2.

• Mop a pre-determined floor area with the mop.

• Rinse the used mop in the bucket containing rinsing water –

Bucket 3.
 Choosing, Using, and Handling Cleaning Material 455

• Wring the mop to remove rinsing water into the waste bucket –
Bucket 2.

• Soak the mop in the cleaning solution in Bucket 1.

• Wring the mop to remove excess cleaning solution into the waste
bucket – Bucket 2.

• Mop a pre-determined floor area with the mop.

• Repeat the process.

Advantage of the two-bucket system is that one and the same mop
can be used for quite a large floor area. The three-bucket system is
much safer, as compared to the two-bucket system since a rinsing
step is added. This rinsing step minimizes the risk of distributing
contaminants release from one floor area to another.

Figure 6 The three-bucket system process

A B

Source: Author

Pre-wetted single-use mops

Comparing the three different systems using re-usable, washable
mops, the more buckets the better cleaning. However, these systems
all include hardware in the form of buckets, often also some type of
456 Cleanroom Contamination Prevention and Control

bucket trolley, and a mop handle and head. From a practical point
of view there is often a desire to minimize as much hardware as
possible. The reason for this is that, for example, a bucket trolley is
quite big and hard, not to say impossible, to clean to the appropriate
cleanliness level. The same aspect applies for all the different buckets
used. This is one of the reasons why single-use and pre-wetted mops
are used to a higher extent today (see Figure 7).

Figure 7 Single use mop

Source: Courtesy of Benchmark

Products

When setting a cleaning program there is a need to determine what

is to be cleaned, how to clean as well as choosing the most practical
solution for each cleanroom, cleanroom airlock, etc. Factors as time,
cost, safety for the cleaning operators, surface area to be cleaned,
storage capacity of clean mops, storage capacity of used mops to be
sent for cleaning, etc., play important roles when determining what
cleaning system to use.
 Choosing, Using, and Handling Cleaning Material 457

SUMMARY
Cleaning of cleanroom, air locks, tabletops, process equipment,
etc. is of vital importance in order to obtain and maintain the
correct and desired cleanliness. Cleaning is, quite contradictive,
one of the most contaminating activities is a cleanroom. Cleaning
a cleanroom is a delicate matter – it must be performed in a precise
manner following logical principles, since it is impossible to judge if
a surface has been cleaned or not, just by visual inspection.

The choice of cleaning material is also of vast importance –

the chosen material must be able to release and capture what is
defined as a contamination and, furthermore, the material should
not release the captured contaminants nor particles arising from the
cleaning material itself. Different cleaning materials, in the form of
whips and mops are available, and it is important to choose material
corresponding to the cleanroom cleanliness class or grade in which
the cleaning is to be performed.

It is important to determine and use cleanroom wipes and mops

which will meet the needs for each cleaning process in a cleanroom.

REFERENCES
EudraLex (2008) The Rules Governing Medicinal Products in the
European Union. Volume 4 – Guidelines for Good Manufacturing
Practices for Medicinal Products for Human and Veterinary Use.
Annex 1 Manufacture of Sterile Medicinal Products. European
Commission, Brussels, Belgium.

ISO (2015) ISO 14644-1: Cleanrooms and Associated Controlled

Environments – Part 1: Classification of Air Cleanliness by Particle
Concentration. International Organization for Standardization
(ISO), Geneva, Switzerland.
458 Cleanroom Contamination Prevention and Control

ISO (2004) ISO 14644-5 Clean Rooms and Associated Controlled

Environments – Part 5: Operations. International Organization
for Standardization (ISO), Geneva, Switzerland.

Ramstorp, M. (2011) Chapter 25 in Microbiology and Sterility Assurance

in Pharmaceuticals and Medical Devices Technology. Eds. Saghee,
M.R., Sandle T., Tidswell E.C. Business Horizons, New Delhi,
India.

Ramstorp, M. (2000) Introduction to Contamination Control and

Cleanroom Technology. Wiley VCH Verlag GmbH, Weinheim,
Germany.

Sandle, T. (2010) Cleaning Cleanrooms. Cleanroom Technology 18(12):

22–24.

ABOUT THE AUTHOR

Matts Ramstorp has a master’s degree in chemical engineering from
LTH, University of Lund, Sweden. He continued his studies and
received his Doctoral Degree in Pure and Applied Biochemistry at
the same university. Matts is the founder of BioTekPro AB. Matts
also worked intensely with R3Nordic, the contamination control
society of the Nordic countries. During this period, he was the chief
secretary and later general secretary of the R3Nordic. He was the
president of ICCCS, International Confederation of Contamination
Control Societies, from 1998 to 2000. He was also the first professor
in cleanroom technology in Sweden at the Design Centre, LTH,
University of Lund.

Matts Ramstorp has at present written more than 10 different

textbooks in the area of contamination control and cleanroom
technology. He is a well-known lecturer within the industrial as well
as the academic world and a member of PHSS, Pharmaceutical and
Healthcare Sciences Society, and has through his role as a member
in SIG, the Special Interest Group, been an active part of PHSS
commenting on the draft Annex 1 (2017), Eudralex, Volume 4.
 INDEX

air changes 85, 105, 112, 172, 237, barrier technology 102
239, 255, 256, 257, 258, 261, 268, bioburden 13, 35, 38, 40, 110, 163,
272, 273, 420 168, 173, 193, 304, 305, 315, 316,
airborne particle concentration 88, 329, 331, 395, 397, 400, 407, 423,
289, 336 428
airflow direction 148, 167, 289, 292 biocidal products 383, 403
airflow intensity 87, 289 bucket assembly 418, 423
airflow pattern 85, 108, 128, 129, 132,
133, 135, 167, 173, 232, 260, 291 cellulose 6, 421, 448
airflow profiles 140 CEN 389, 401, 402, 403
airflow visualization 64, 128, 130, 173, CFD modeling 132, 133, 136
230, 234, 246, 268, 273, 274 CFD principle 131
ANSI/AAMI/ISO 11737-2: 2009 314 cleaning technique 439, 440
AOAC 387, 388, 389, 390, 391, 392, cleanroom wipes 446, 448, 457
393, 398, 407, 409, 410, 414, 415 clothing 4, 155, 224, 232, 233, 311,
application method 419, 420, 428 321, 322
as built 134, 223, 247, 275, 288, 293, computational fluid dynamics 17, 22,
295, 298 85, 129, 131, 230, 289
at rest 84, 134, 153, 171, 223, 232, conductive materials 204
252, 265, 268, 289, 332, 336, 347, construction documents 119, 120, 121
438, 444 containment leak 239, 290

459
460 Cleanroom Contamination Prevention and Control
contamination risk 13, 43, 51, 67, 107, floors 119, 168, 207, 208, 212, 224,
148, 172, 178, 180, 226, 372 262, 290, 419, 435, 437, 450
corrective and preventive actions
(CAPA) 2, 15 gamma irradiation 308, 313, 447
critical process 390, 438 garment style 309
gown room 136, 310, 311, 312, 373,
data integrity 2, 15, 164, 166 379
design considerations 80 grounding 11, 205, 206, 207, 208, 209,
design development 119, 120 212, 215, 310
Design Qualification (DQ) 80, 81, 132,
168, 235, 236, 293 HEPA filter 18, 19, 22, 23, 99, 109,
design review 123, 124, 288 154, 156, 165, 179, 184, 250, 252,
deviant air 26, 151, 154, 155, 157, 162, 253, 260, 275, 290
166, 178 HVAC system 9, 82, 88, 89, 99, 100,
disinfectant efficacy 384, 389, 397, 400, 109, 110, 112, 128, 136, 226, 227,
423, 424, 425 238, 239, 247, 274, 296
disinfectant validation 384, 385, 396,
399, IEST-RP-CC003.3 313
dispersion 62, 63, 134, 191, 243, 360, in operation 153, 171, 223, 275, 332,
439 338, 347
dissipative material 205, 207, 208 induction 151, 203, 225, 255
Installation Qualification (IQ) 80, 81,
eddy currents 22, 23, 26, 151, 155, 235, 237, 247, 293, 294, 297, 298,
157, 162, 163, 164, 170, 178, 179, insulators 199, 200, 203, 204, 205, 206,
184, 186 207, 210, 211, 212
electrostatic forces 197, 198, 199, 369,
437 knowledge 2, 24, 39, 50, 52, 56, 65,
electrostatic discharge 79, 201, 206 66, 69, 70, 72, 278, 441, 443
EN 13697 387, 391, 394, 398, 405,
406, 407 laundering process 304, 311
EN 1500: 2013 402 lessons learned 11, 13, 15, 17, 20, 22,
energy saving 90, 127, 275 23, 25, 26, 28, 29, 31, 32, 33, 34,
35, 36, 38, 39, 40, 41, 42
fabric 210, 303, 304, 305, 306, 307,
308, 310, 313, 320 managing risk 69
Factory Acceptance Testing (FAT) 21, microbial contamination 2, 3, 8, 9, 16,
22, 152, 160, 178, 179, 183, 236, 34, 62, 63, 148, 171, 219, 242,
288 276, 298, 329, 332, 400, 418, 426
first air concept 154
 Index 461
non-unidirectional 10, 85, 146, 148, review outcome 124
151, 153, 155, 177, 167, 191, 289 risk identification 52, 58, 72
risk ranking 56, 61, 64, 65
Operational Qualification (OQ) 80, 81, risk review 69
223, 235, 238, 239, 293, 296, 298 risk to patient 5, 26, 52, 53, 63, 65, 69
operator behavior 181, 440, 443, 444 robotic 103, 205, 207

particle deposition 199, 239, 291 schematic design 119, 120

particle migration 93, 94, 136, 139, seams 290, 307, 308, 309, 377
291, 292, 297, 304 single use 303, 304, 305, 306, 307, 308,
PDA Technical Report #70 396 309, 310, 319, 320, 370, 418, 445,
Performance Qualification (PQ) 80, 81, 447, 450, 451, 455, 456
220, 223, 235, 239, 243, 245, 270, smoke study 13, 19, 24, 25, 34, 147,
293, 298 150, 163, 164, 165, 166, 168, 174,
performance testing 288, 289, 296, 175, 176, 177, 184, 186, 192, 193,
306 230, 268
phenolic 30, 31, 368, 369, 423, 426, sticky mats 370, 372, 379
427 structural assembly 378
placement 20, 93, 98, 136, 161, 169, suspension tests 385, 386, 393, 403,
174, 180, 337, 370, 371, 373, 374, 405, 406
375,
polyester 304, 305, 309, 311, 446, 448 test surfaces 388, 390
polymer flooring system 372, 374, 375, traffic 85, 94, 201, 354, 367, 368, 369,
376, 378 371, 372, 373, 375, 377, 423
pressurization 79, 81, 82, 88, 93, 94, turbulence 22, 23, 24, 131, 151, 155,
96, 97, 100, 112, 130, 146, 151, 157, 162, 163, 164, 166, 169, 170,
191, 228, 229, 254, 298, 299 173, 175, 176, 178, 179, 184, 186,
problems 21, 58, 99, 163, 183, 185, 225, 260
262, 297, 299, 357, 417, 428
use dilution 385, 386, 387, 407, 408,
recalls 2, 6, 9, 13, 34, 39, 212 417, 418, 419, 422
recovery time 134, 223, 290, 444, 445 User Requirements Specification (URS)
regulatory observations 2, 6 22, 23, 25, 118, 152, 183, 235,
relative humidity 97, 98, 113, 129, 203, 236
231, 238, 237, 255, 290, 350, 420 USP <1116> 64, 172, 228, 396,
requalification 160, 299 utilities 9, 292, 295, 299, 401
residues 35, 242, 399, 400, 421, 425,
426, 427 Validation Master Plan (VMP) 235, 385
review mechanism 123
462 Cleanroom Contamination Prevention and Control
walls 11, 29, 36, 98, 107, 109, 113,
137, 140, 168, 206, 207, 208,
224, 226, 323, 419, 437, 445, 450
washable 447, 455
Suite 600, Bethesda, MD 20814.