Purification of Monoclonal Antibodies

Purification of MAbs was conducted as described previously (30 (link), 34 (link)). Briefly, positive hybridoma clones were expanded and, when at approximately 12 million cells per culture, ∼8 million cells were frozen in a 1-ml cryostock each. Remaining cells were slowly expanded to large culture volumes (∼800 ml each) in hybridoma serum-free medium (Gibco), from which supernatants were harvested via low-speed centrifugation and sterile filtered via 0.22-μM membranes (EMD Millipore). These supernatants were then affinity purified via gravity flow with protein G-linked Sepharose 4 fast flow beads packed into columns (GE Healthcare). After washing the beads with 3 column volumes (∼500 ml) of sterile PBS (pH 7.4), an elution step was carried out with 45 ml of 0.1 M glycine-HCl buffer (pH 2.7). The eluate was then immediately neutralized with 5 ml of 2 M Tris-HCl buffer (pH 10) to bring the overall solution to a pH of approximately 7.0. The MAbs were then buffer exchanged to PBS (pH 7.4) using 30 kDa Amicon centrifugation filters (EMD Millipore) and washed three times with PBS on the membrane. Finally, the concentration of antibody was quantified using a Nanodrop spectrophotometer (Thermo Scientific), measuring absorbance using the protein A₂₈₀ protocol.