Secreted protein expression and purification
The 10H2 and BS2 antibodies (Patent No. WO/2020/243489, 2020) were expressed via transient co-transfection of gWiz plasmids (Genlantis) encoding their respective heavy and light chains (1:1 ratio) in HEK cells (2 µg DNA/mL culture). The 602 antibody (Patent No. WO/2020/264321, 2020) was expressed in CHO cells (4 µg DNA/mL culture). The optimized DNA concentration for transfection was determined by dose titration in small-scale transfections by adding 4-4-6- and PEI-based nanoparticles (formulated as described above) to 2 mL cells in 6-well plate format. Cells were incubated as described above for 96 to 120 h. Cell supernatants were then incubated with Protein G agarose beads (Thermo Fisher Scientific) for 3 hours at room temperature. Beads were washed and bound protein was eluted using 0.1M glycine, pH 2, and measured by SDS-PAGE analysis. Band quantification was performed using ImageJ software. For larger scale (>2 mL) transfections, 4-4-6- and PEI-based nanoparticles were formed as described above, added to cells in a shaking flask, and incubated as described above for 96 to 120 h. Cell supernatants were then incubated with Protein G agarose beads either at room temperature for 3 hours or at 4°C overnight. Secreted protein was harvested from cell supernatant by Protein G affinity chromatography, and 10H2 and BS2 samples were further purified using a Superdex 200 sizing column equilibrated in PBS on a fast protein liquid chromatography (FPLC) instrument (Cytiva). Yield was quantified by measuring absorbance at 280 nm using a Nanodrop spectrophotometer (Thermo Fisher Scientific).