Superdex 200 increase size exclusion chromatography

As reported previously^{51 (link)–53 (link)}, the IL-10/Fc fusion protein containing a human IL-10 fused at the N-terminal with a noncytolytic human IgG1 Fc was expressed by FreeStyle 293-F Cells (Gibco / Thermo Fisher Scientific) at the EPFL Protein Expression Core Facility. Supernatant of culture medium containing IL-10/Fc fusion protein was harvested by centrifugation after a 7-day culture and was filtered through a 0.22-μm membrane to obtain a clear solution. The recombinant protein was first captured with a HiTrap Protein A affinity chromatography column on an AKTA pure 25 (GE Healthcare), and eluted with an elution buffer (0.05-M sodium citrate, 0.3-M sodium chloride, pH = 3.0). The eluted protein was collected immediately in a neutralization buffer (1-M Tris–HCl, pH = 10.0) followed by concentration with membrane ultrafiltration (molecular weight cut-off 10 kDa) in a Vivaspin (GE Healthcare). The concentrated protein solution was further purified with a Superdex 200 increase size exclusion chromatography (GE Healthcare) at a flow rate of 1.0 mL/min with phosphate buffered saline (PBS) buffer on AKTA pure 25 (Extended Data Fig. 1a,b). The purified protein was aliquoted and stored at -80 °C before use. The purity of IL-10/Fc was confirmed with sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE, Extended Data Fig. 1c).