Disulfide bond reduction of antibodies during clarification: A bench‐scale assay and a mitigation tool

We investigated side-by-side clarification with a two-stage depth filter system against a single-stage charge chromatographic clarifier on a number of Chinese hamster ovary (CHO) cell cultures. We found that the one-stage fibrous anion exchange (AEX) clarification met or exceeded the performance of the conventional two-stage depth filtration systems in terms of product recovery, removal of process-related impurities, and throughput. The one-stage anion clarification approach had a smaller footprint, a shorter set-up time, an easier setup, consumed less buffer flush volume, and operated at a lower differential pressure relative to those of conventional systems. In a comparability study at 200 L scale, antibodies in cell culture fluid (CCF) clarified by depth filtration system exhibited product quality degradation due to disulfide bond reduction (DSBR), whereas antibodies clarified by AEX fiber method were not. We surmised that the lower operating differential pressure and electrostatic adsorption mechanism of the anion clarification system minimized the potential of product to undergo DSBR. We developed a bench-scale process model that allowed us to assay DSBR and predict the process results. We then utilized it to demonstrate that the antibody was reduced when clarified with a two-stage 3 M™ Zeta Plus™ Depth Filter system but not with the anionic clarification system. This bench-scale DSBR assay allowed us to rank the relative disulfide bond reduction potentials of various monoclonal antibodies (mAbs) and demonstrate the reduction-mitigating impacts of the fibrous clarification approach on a reduction-prone antibody culture at 2000 L processing scale.