Antibody expression and purification

FreeStyle™ 293-F cells (Thermo Fisher Scientific, Cat#{"type":"entrez-nucleotide","attrs":{"text":"R79007","term_id":"855288","term_text":"R79007"}}R79007) were cultured in serum-free medium. Transient transfection was performed by co-transfection of expression vectors encoding a heavy chain or a light chain individually into FreeStyle™ HEK293-F cells using 1 μg/ml 25 KDa linear polyethylenimine (Polysciences, Inc.). One day after transfection, valproic acid (Sigma) was added to cell culture at a final concentration of 3 mM. On day 2 post-transfection, medium comprising 10% GlutaMAX, 10% 400 g/L glucose and 80% freestyle 293 medium was added to the cell culture at 10% of the total volume. Conditioned medium was collected 5–6 days after transient transfection. Antibodies in the culture media were purified by MabSelect SuRe affinity columns (GE Healthcare) on an AKTA Avant 25 fast protein liquid chromatography (FPLC) System. The columns were equilibrated with buffer A (25 mM sodium phosphate, 150 mM sodium chloride, pH = 7.0) prior to use. The culture media containing antibodies were then applied to the columns followed by elution with Buffer B (100 mM sodium citrate, pH 3.5) to collect the desired proteins. Collected proteins were neutralized with 1 M Tris (pH 9.0), which were then dialyzed against phosphate-buffered saline (PBS). Finally, the purity of the samples was analyzed on a SEC-high performance liquid chromatography (SEC-HPLC).