Protein a resin

Antibodies and Fab fragments were produced as previously described^{17 (link)}. Briefly, heavy and light chain plasmids (IgG format) containing secretion signals were co-transfected in Expi293F cells (ThermoFisher) using Turbo293 transfection reagent (Speed Biosystem). Cells were incubated for 1 day at 37 °C, followed by 4 days at 37 °C. All subsequent steps were performed at 4 °C. Supernatant was collected by centrifugation and loaded onto Protein A resin (GE Healthcare) pre-equilibrated with PBS. Bound antibodies were washed with 50 ml of PBS and eluted dropwise in 1 mL fractions with Pierce IgG Elution buffer (Pierce). Elution was neutralized with 1 M Tris-Cl, pH 8.0 (final concentration 0.1 M). Fractions with highest A₂₈₀ absorption were pooled and dialyzed overnight against PBS. Dialyzed protein was concentrated to ~10 mg/mL, filter sterilized, and kept at 4 °C until needed. For the production of Fab fragment, the purified antibodies were incubated with HRV-3C protease (Millipore-Sigma) overnight at 4 °C. Cleavage reaction was loaded onto Protein A resin (GE Healthcare), and flow-through was collected. Fabs were purified by size-exclusion chromatography on a Superdex 200 16/60 column in PBS. Fractions corresponding to Fab were pooled, concentrated to ~5 mg/mL, filter sterilized, and kept at 4 °C until needed.

All human monoclonal antibodies were produced according to the procedure reported previously (Anami et al., 2017 (link); Shi et al., 2014 (link)). Briefly, free style HEK-293 human embryonic kidney cells (Invitrogen) were transfected with a mammalian expression vector encoding for the human IgG1 kappa light chain and full-length heavy chain sequences (based on variable sequences of cetuximab, depatuxizumab, ortrastuzumab). The transfected HEK-293 cells were cultured in a humidified cell culture incubator at 37°C with 8% CO₂ and shaking at 150 rpm for 7 days before harvesting the culture medium. The antibody secreted into the culture medium was purified using Protein A resin (GE Healthcare).

Candidate antibodies were expressed with Expi-CHO Expression system(gibco) for 12days and the supernatant was harvested and purified by protein A resin (GE healthcare). The antibodies were further purified by Q FF (GE healthcare) and Capto S ImpAct (GE healthcare) sequentially and then changed to buffer containing 10 mM CH₃COONa·3H₂O, 30 mM NaCl, 5% sucrose, 0.03% tween-20, pH6.0.