Tutorial 2
Q1. Tubes of 0.1 cm diameter (Dh) and 100 cm length are used as flow channels in an
ultrafiltration system. The system can operate at a bulk stream velocity of 300 cm/s. At this
velocity, determine the polarization modulus for a transmembrane flux of 45 L/m2h.
Diffusivity of protein (A) in the solution (B) = 9 X 10-7 cm2/s.
Viscosity of solution= 1.2 cP
Density of solution = 1.1 g/cm3
Mass transfer coefficient, k = [Link]/Dh
Sherwood number, Sh = 0.082 Re0.69 Sc0.33
Q2. Calculate the TFF membrane area for concentrating 100 L protein sample to 10 L in 3.5
hours assuming flux to be 100 L/m2/h.
Q3.
i) Find the area of a tangential flow filtration (TFF) system to be used to concentrate a 10 L
to 200 mL in 2.5 hrs? Assume the average filtrate flux rate of 50 LMH.
ii) A sample with 0.5M NaCl is resuspended in 50 mL. The salt concentration needs to be
lowered to less than 0.05M before being concentrated to 10 mL. How long will it take a
50 cm3 TFF capsule with the suitable cut size, if the average filtrate flux rate is 40 LMH and
3 diafiltration volumes are needed to lower the salt concentration to the desired 0.05M?
Q4. Given the following chromatogram for the separation of two solutes on a 25 cm long
Size Exclusion Chromatography (SEC)-HPLC column, determine:
a) HETP achieved under the operating
conditions
b) the resolution achieved for solutes A and B
c) the capacity factors for solutes A and B.
d) why would A & B have different
retention times?
Assume that the exact retention time for the
compound A (tRA ) is 6.0 min and B tRB = 7.8
min and that the width of the eluting peak at
the baseline for compound A is 1.5 min and
for compound B is 1.95 min. Also, the
column yielding a tm = 0.9 min.
Q5. A lab treats 200 L of cell broth containing 10 g/L of protein G CSF to recover and purify
the product. The impurities are removed from the broth centrifugation with a product yield of
75%. Host Cell Proteins (HCPs) are then removed by salt precipitation with a product yield
of 60% for these techniques. The clear GCSF fraction is treated further using gel
chromatography and affinity chromatography; 85% yield is achieved in each of the
chromatography steps.
a) What is the overall product yield?
b) How much purified G CSF is produced overall?
c) It is decided to improve the salt precipitation step to minimise product
loss. If the target G CSF production rate is 8 kg/cycle from a 200 L of broth, by
how much must the product yield for this stage of the process be increased above the
current output?
Q6. Gel chromatography is used for commercial-scale purification of the proteinaceous
diphtheria toxoid from Corynebacterium diphtheriae supernatant. In a laboratory, a
small column of 1.5 cm inner diameter and height 0.4 m is packed with 10 g dry
Sephadex gel; with a void volume of 23.0 mL. A sample containing the toxoid and
impurities are injected into the column, at a flow rate of 14 mL/min, the elution volume for the
toxoid is 29.0 ml and the elution volume for the principal impurity is 45 ml.
A column of height 0.6 m and diameter 0.5 m is available for large-scale gel
chromatography. The same type of packing is used; the void fraction and the ratio of
pore volume to total bed volume remains the same as in the laboratory-scale column.
The liquid flow rate in the large column is scaled up in proportion to the column cross-
sectional area; the flow patterns in both columns can be assumed identical. The water regain
value for the packing is given by the manufacturer as 0.0035 m3/kg dry gel.
(a) Determine the gel partition coefficients for the toxoid and impurity.
(b) Calculate the elution volumes in the commercial-scale column.
(c) What would be the volumetric flow rate in the large column?
(d) Estimate the retention time of toxoid in the large column.
Q7. At pH 7.0, in what order would the following three peptides be eluted from a column
filled with a cation-exchange polymer? Their amino acid compositions are:
Protein A: Ala 10%, Glu 5%, Ser 5%, Leu 10%, Arg 10%, His 5%, Ile 10%, Phe 5%, Tyr
5%, Lys 10%, Gly 10%, Pro 5%, and Trp 10%.
Protein B: Ala 5%, Val 5%, Gly 10%, Asp 5%, Leu 5%, Arg 5%, Ile 5%, Phe 5%, Tyr 5%,
Lys 5%, Trp 5%, Ser 5%, Thr 5%, Glu 5%, Asn 5%, Pro 10%, Met 5%, and Cys 5%.
Protein C: Ala 10%, Glu 10%, Gly 5%, Leu 5%, Asp 10%, Arg 5%, Met 5%, Cys 5%, Tyr
5%, Phe 5%, His 5%, Val 5%, Pro 5%, Thr 5%, Ser 5%, Asn 5%, and Gln 5%.
