Separation of recombinant monoclonal antibodies IgG201 from a cell culture supernatant using an integrated aqueous two-phase system with thermo-separating EOPO

Abstract The purification of monoclonal antibodies IgG201 produced in Chinese hamster ovary cells was accomplished by aqueous two-phase system (ATPS) extraction. Host cell proteins, pigments, and other impurities were removed during ATPS extraction. Two simulation modules, Blends and Dissipative particle dynamics (DPD), from Material Studio software, were innovatively applied to investigate the mechanisms of antibody extraction using ATPS. The simulation results indicated that antibodies were extracted from the cell culture supernatant into the EOPO polymer phase at a pH close to or higher than its isoelectric point, while antibodies were transferred from the EOPO phase to the water phase at a pH much lower than its isoelectric point. In addition, the feasibility of using an EOPO polymer to extract monoclonal antibodies from a cell culture supernatant was investigated at different pH, compositions, and EOPO polymer contents. In the forward-extraction ATPS, a monoclonal antibody was extracted from the cell culture supernatant to the EO20PO80 polymer phase at pH 9. In the subsequent back-extraction ATPS, the monoclonal antibody was transferred to the water phase at pH 5. An integrated purification process combined with two-step ATPS and chromatography was applied to antibody IgG201. Under optimal conditions of 20% EO20PO80 at pH 9 in forward extraction and 33% EO20PO80 at pH 5 in back extraction, a total recovery of 93.12 ± 0.77% and purity of 90.29 ± 0.07% were obtained. With the integration of cation exchange (CEX) chromatography, a final purity of 98.55 ± 0.15% with a recovery of 75.18 ± 2.33% was obtained. Thus, the ATPS-CEX workflow achieved comparable recovery and purity with traditional protein A based workflow.