Tailoring polishing steps for effective removal of polysorbate‐degrading host cell proteins in antibody purification

Ensuring the quality and safety of biopharmaceutical products requires the effective separation of monoclonal antibodies (mAbs) from host cell proteins (HCPs). A major challenge in this field is the enzymatic hydrolysis of polysorbates (PS) in drug products. This study addresses this issue by investigating the removal of polysorbate‐degrading HCPs during the polishing steps of downstream purification, an area where knowledge about individual HCP behavior is still limited. We investigated the separation of different mAb formats from four individual polysorbate degrading hydrolases (CES1F, CES2C, LPLA2, and PAF‐AH) using cation exchange (CEX) and mixed‐mode chromatography (MMC) polishing steps. Our research identified a key challenge: The similar elution behavior of mAbs and HCPs during chromatographic separation. To investigate this phenomenon, we performed high‐throughput binding screenings for recombinant polysorbate degrading hydrolases and representative mAb candidates on CEX and MMC chromatography resins. We then employed a three‐step strategy that also served as a scale‐up process, optimizing separation conditions and leading to the successful removal of specific HCPs while maintaining high mAb recovery rates (>96%). This strategy involved the use of surface response models and miniature columns for screening, followed by validation on larger columns using a chromatography system. Our results highlight the critical role of the inherent properties of mAbs for successful separation from HCPs. These results underscore the need to tailor the purification process to leverage the slight differences in binding behavior and elution profiles between mAbs and specific HCPs. This approach lays the foundation for developing more effective strategies for overcoming the challenge of enzymatic polysorbate degradation, paving the way for improved quality and safety in biopharmaceutical products.