Hitrap protein a hp column

Recombinant chimeric antibodies were produced using Expi293 Expression System (Thermo Fisher Scientific) following the manufacturer’s instructions. Briefly, Expi293F cells were transfected with the vectors encoding the heavy and light chains (30 μg each) per cell culture of 60 mL using the ExpiFectamine 293 Transfection Kit (Thermo Fisher Scientific). The cell culture was incubated on a shaker for 6–7 days post transfection, and the cells were removed by centrifugation at 6,000 × g for 20 min. The culture supernatant was filtered through a 0.20 μm filter, and the antibodies were captured in a 1 mL HiTrap Protein A HP Column (Cytiva). The column was washed with 20 mL phosphate-buffered saline (PBS), and the antibodies were subsequently eluted with 5 mL citrate buffer (100 mM, pH 3.0). The eluate was dialyzed in PBS and used without further purification.

IgG1 Fcs were expressed from a pFUSE-hIgG1-Fc vector (encoding residues 221-447 of IgG1; plasmid purchased from InvivoGen). Wild-type IgG1 Fc was expressed exactly as encoded within this plasmid; mutations for the E382A/S/R constructs were introduced by site-directed mutagenesis (QuikChange II kit; Agilent), using mutagenic primers (Supplementary Table 4) synthesised by Eurofins Genomics. Sequences of resulting IgG1 Fc constructs are shown in Supplementary Fig. 11. Fcs were transiently expressed in FreeStyle293F cells (ThermoFisher), using FreeStyle™ MAX Reagent (ThermoFisher), as described in the manufacturer’s protocol. Cells were left to incubate at 37 °C, 8% CO₂, shaking at 125 rpm (New Brunswick S41i incubator), and harvested after seven days by centrifugation at 3100 x g for 30 minutes. Supernatants were filtered through a 0.2 μm membrane and antibodies purified by affinity purification with a HiTrap Protein A HP column (Cytiva), followed by size exclusion chromatography with a Superdex 200 16/600 column (Cytiva) in 10 mM HEPES, 150 mM NaCl (pH 8.0).

The pFUSE-fPD1-hIg#1 and pFUSE-fPDL1-hIg#1 were transduced into Expi293 F cells using the Expi293 expression system (Thermo Fisher Scientific, Waltham, MA, USA) according to manufactures’ instruction. fPD1-hIg and fPD-L1-hIg were collected and purified using a HiTrap Protein A HP column (Cytiva) and desalted using a PD10 column (Cytiva)^{24 (link)}.