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Affinity Processing of Cell-Containing Feeds 251
3.2. Coupling Affinity Ligand: Protein A
1. Connect 2-ml cryogel column to a pump and wash with 20 ml of water at a flow
rate of 1 ml/min and then with 0.2 M Na 2 CO 3 (20 ml).
2. Apply ethylenediamine solution (0.5 M in 0.2 M Na 2 CO 3 ; 30 ml) to the column
at a flow rate of 75 cm/h in recycle mode for 4 h.
3. Wash with water until pH is close to neutral.
4. Wash with 20 ml, 0.1 M sodium phosphate buffer, pH 7.2.
5. Apply glutaraldehyde solution (5% v/v; in 0.1 M sodium phosphate buffer, pH 7.2,
30 ml) to the column at a flow rate of 75 cm/h in recycle mode for 5 h.
6. Recycle protein A solution (1.6 mg/ml; in 0.1 M sodium phosphate buffer, pH 7.2,
12 ml) through the column at a flow rate of 75 cm/h at 4°C for 24 h.
7. Apply the freshly prepared NaBH 4 solution (0.1 M in sodium carbonate buffer,
pH 9.2, 30 ml) to the column at a flow rate of 75 cm/h for 3hinrecycle mode
to reduce Schiff base formed between the protein and the aldehyde-containing
matrix.
8. The amount of protein A immobilized on polyDMAAm monolithic cryogel matrix
is determined by the BCA method according to a modified method given by Smith
et al. (12). A suitable amount of dried protein A cryogel pieces are well suspended
in water by finely grinding and ultrasonication. To different amounts of the protein
A gel suspension (20–100 μl) is added 2 ml of the BCA solution, and the mixture is
incubated at 37°C with thorough shaking for 30 min. The absorbance is measured
at 562 nm. Appropriate controls are taken using native poly DMAAm cryogel.
The standard curve is made by quantitative additions of the protein A to the native
polyDMAAm cryogel and absorbance measured under the same conditions.
3.3. Direct Capture of (His) 6 -Tagged Single-Chain Fv Antibody
Fragments (See Note 1)
1. Wash IDA-cryogel column with 4 CV of distilled water, followed by 4 CV of 0.25
M CuSO 4 in distilled water and finally by 4 CV of distilled water (see Note 2).
2. Equilibrate column with 5 CV of 20 mM HEPES, 200 mM NaCl, 2 mM imidazole,
pH7(see Note 3).
3. Load 1-ml sample containing non-diluted cell culture fluid containing 24–32 μg/ml
His single-chain Fv at a flow rate of 300 cm/h (see Note 4).
4. Wash with 5 CV of 20 mM HEPES, 200 mM NaCl, 2 mM imidazole, pH 7, at a
flow rate of 300 cm/h (see Note 5).
5. Elute with 0.2 M imidazole in 20 mM HEPES, 200 mM NaCl, pH 7, at a flow
rate of 300 cm/h (see Note 6).
6. Regenerate the column with 10 CV of 20 mM EDTA in 20 mM HEPES, 200
mM NaCl, pH 7. Store column at 4°C, preferably in the presence of antimicrobial
agent (see Note 7).
7. Analyze eluted fractions for protein content, for example, using BCA assay
according to ref. 12, and for the content of target protein (see Note 8).