Freestyle 293 serum free expression medium

Two plasmids that included coding sequences for full-length IgG1 heavy and light chains were transfected into HEK293-H cells using polyethyleneimine (PEI). Five hundred micrograms PEI were incubated for 25 min at room temperature with 250μg of each plasmid and then added to the HEK293-H at a density of 1×10⁶ cells/ml in total volume of 500ml (29 (link), 30 (link)). Transfected cells were adapted for growth in Freestyle 293 serum-free expression medium (Gibco, Thermo Fisher Scientific) under suspension conditions of 37°C and 8.5% CO2. Cells were centrifuged 96 h post transfection and culture medium was harvested, filtered through a 0.22 μm filter and supernatants concentrated 20 times using a 50 kDa cut-off Vivaflow 50 System (Vivasciences). One volume of IgG binding buffer (Thermo Scientific Pierce) was added and IgG was purified on a Protein A HP HiTrap column (GE Healthcare) eluted with IgG elution buffer (Thermo Scientific Pierce) and neutralized with 1M Tris pH 9.0. Proteins were concentrated and buffer exchanged with PBS using Amicon Ultra4 10kDa. Protein concentrations were determined on Nanodrop (Thermo Fisher Scientific) and sample purity was analyzed by SDS-PAGE.

Two plasmids that included coding sequences for full-length IgG1 heavy and light chains were transfected into HEK293-H cells using polyethyleneimine (PEI). Five hundred micrograms PEI were incubated for 25 min at room temperature with 250μg of each plasmid and then added to the HEK293-H at a density of 1×10⁶ cells/ml in total volume of 500ml^{54 (link),55 (link)}. Transfected cells were adapted for growth in Freestyle 293 serum-free expression medium (Gibco, Thermo Fisher Scientific) under suspension conditions of 37°C and 8.5% CO2. Cells were centrifuged 96 h post transfection and culture medium was harvested, filtered through a 0.22 μm filter and supernatants concentrated 20 times using a 50 kDa cut-off Vivaflow 50 System (Vivasciences). One volume of IgG binding buffer (Thermo Scientific Pierce) was added and IgG was purified on a Protein A HP HiTrap column (GE Healthcare) eluted with IgG elution buffer (Thermo Scientific Pierce) and neutralized with 1M Tris pH 9.0. Proteins were concentrated and buffer exchanged with PBS using Amicon Ultra4 10kDa. Protein concentrations were determined on Nanodrop (Thermo Fisher Scientific) and sample purity was analyzed by SDS-PAGE.