Protein a sepharose 4 fast flow chromatography

The development of antibodies 3.3 and 2B5 have been described previously [7 (link), 13 (link)]. 2.5 × 10⁷ of 3.3 or 2B5 hybridoma cells in 15 mL culture medium (DMEM, 5% FBS) were inoculated into a CELLine CL 1000 two-compartment bioreactor (INTEGRA Biosciences AG). The antibody-containing culture medium was harvested every 7 days and then purified by protein A Sepharose 4 Fast Flow chromatography (GE Healthcare). The collected antibody solution was dialyzed against PBS and sterile filtered. Antibody concentrations were determined by the bicinchoninic acid (BCA) protein assay (Thermo Scientific).

293 T cells (1.5 × 10⁶ cells/well) were seeded in 6-well plate contained in DMEM supplemented with 10% cosmic calf serum and placed in an incubator at 37 °C overnight. The cells were transfected with 2.5 μg pLHCX-pro-anti-EGFR or pLHCX-anti-EGFR plasmids by TransIT-LT1 transfection reagent (Mirus Bio Corporation, Madison, WI, USA) and cultured in DMEM serum-free medium for 48 hours. The cells and media were collected for subsequent experiments. Alternatively, 6 × 10⁷ Expi293 cells in suspension were transfected with 30 μg pLHCX-LAP-anti-TNF-α or pLHCX-anti-TNF-α plasmids by the Expifectamine reagent (Life Technologies, Carlsbad, CA, USA). Cells were cultured in Expi293 media with enhancers as specified by the manufacturer. The media were harvested for subsequent experiments 5 days post-transfection. The anti-EGFR antibodies and LAP-anti-EGFR antibodies were purified by protein A sepharose 4 Fast Flow chromatography (GE Healthcare, Little Chalfont, UK). The purity of purified anti-EGFR antibodies and LAP-anti-EGFR antibodies were analyzed by SDS-PAGE in reducing and nonreducing conditions (supplementary Figure 4).