IB5751 Downstream Processing: Assignment II
Submission due on 12 August, 2025
1. The following is the data for concentration of BSA in an aqueous solution at pH 4.8 and the recorded osmotic pressure
in an acetate buffer at 0oC. Determine the molecular weight of BSA.
BSA conc, g/ml Osmotic pressure π (cm
H2O)
0.78 2.39
1.25 4.01
2.79 8.53
3.38 10.8
4.18 13.01
8.98 27.84
12.45 37.69
2. Determine the molecular weight and the osmotic virial coefficient of α-chymotrypsin in water at pH 4.0 in an 0.01 M
potassium sulfate solution at 25oC.
Cs (g/l) Osmotic pressure π (Pa) π/Cs (Pa.L/g)
0.902 74.8 82.92
1.798 142.3 79.14
3.574 276.2 77.29
5.331 398.0 74.65
7.027 513.3 73.05
8.835 625.3 70.77
Use these data to determine the molecular weight of α-chymotrypsin and the value of its osmotic second virial coefficient
B2 at these conditions
3. Determine the diffusivities and approximate molecular sizes at 25 oC of following biological materials in aqueous
solutions using theoretical correlations you have learnt
Biological material MW, Da Biological material MW, Da
Alcohol dehydrogenase 79,070 Soybean protein 361,800
Aprotinin 6,670 Trypsin 23,890
Bovine serum albumin 67,500 Urease 482,700
Cytochrome C 11,990 Ribonuclase A 13,690
g–Globulin, human 153,100 Collagen 345,000
Hemoglobin 62,300 Fibrinogen, human 40,600,000
Lysozyme 13,800 Lipoxygenase 97,440
Tobacco mosaic virus 40,600,000 Alcohol oxidase 675 000
4. Current and future pharmaceutical products of biotechnology include proteins, nucleic acids, and viral gene vectors.
Identify five physical and biochemical features of these biological species by which they could be distinguished in a
bioseparation; Identify a bioseparation operation that could be used to selectively remove or retain each species from a
mixture of the other two; and summarize important considerations in maintaining the activity of each species that would
constrain the operating parameters of each bioseparation. Extend that example by listing the purity requirements for FDA
approval of each of these three purified species as a parenteral product, which is one that is introduced into a human
organism by intravenous, subcutaneous, intramuscular, or intramedullary injection.
5. Separation processes for bioproducts from E. coli. Recombinant protein production from E. coli resulted in the first
products from biotechnology. (a) List the primary structures and components of E. coli that must be removed from a
fermentation broth to purify a heterologous protein product (one that differs from any protein normally found in the
organism in question) expressed for pharmaceutical use. (b) Identify a sequence of steps to purify a conjugate
heterologous protein (a compound comprised of a protein molecule and an attached non-protein prosthetic group such as a
carbohydrate) that remained soluble in cell paste. (c) Identify a separation operation for each step in the process and list
one alternative for each step. (d) Summarize important considerations in establishing operating procedures to preserve the
activity of the protein. (e) Suppose net yield in each step in your process was 80%. Determine the overall yield of the
process and the scale of operation required to produce 100 kg per year of the protein at a titer of 1 g/L.
6. As an example of solubility of weak organic acids, bases, and zwitterions in biological systems, prepare total solubility
curves for the following species across a broad pH range (pH = 1 to pH = 11).
(i) Caprylic acid (C8H16O2) is an oily, naturally occurring liquid in coconuts that has anti-fungal and anti-bacterial
properties. Its water solubility is 0.068 g/100 g at 20oC with a value of pKa = 4.89.
(ii) Thymidine (T, C5H6N2O2, 5-methyluracil) is a pyrimidine base that forms two hydrogen bonds with adenine
(A) to stabilize dsDNA. Its water solubility is 4.0 g/kg at 25oC and its pKBOH is 9.9 (neglect further dissociation
that occurs at higher pH).
(iii) The hydrophobic amino acid valine (C5H11NO2), which substitutes for hydrophilic glutamic acid in
hemoglobin, causes misfolding, which results in sickle-cell anemia. Its water solubility is 8.85 g/100 mL at 25 oC.
Its ionization constants are pKa = 2:3 and pKa= 9:6.
Calculate the solubility of each component in the following bodily fluids: cell cytosol, pH = 7.2; saliva, pH = 6.4; urine,
pH = 5.8.
7. Phosphate-buffered saline (PBS) finds wide usage in biological processing, formulation, and research. Its pKa and ionic
strength mimic physiologic conditions and maintain pH across temperature changes expected in biological systems (4 –
37oC). PBS contains sodium chloride, sodium phosphate, and (in some formulations) potassium chloride and potassium
phosphate at osmolarity (the concentration of osmotically active particles in solution) and ion concentrations that match
those in the human body (isotonic). One common formulation is to prepare 10-liter stock of 10x PBS by dissolving 800 g
NaCl, 20 g KCl, 144 g Na2HPO4.2H2O, and 24 g KH2PO4 in 8 L of distilled water, and topping up to 10 L. Estimate the
pH of this solution in its concentrated form when stored at 4oC, after being diluted to 1x PBS at 4oC, and after being
warmed to 37oC.
