Host cell protein networks as a novel co‐elution mechanism during protein A chromatography

Abstract

AbstractHost cell proteins (HCPs) are process‐related impurities of therapeutic proteins produced in for example, Chinese hamster ovary (CHO) cells. Protein A affinity chromatography is the initial capture step to purify monoclonal antibodies or Fc‐based proteins and is most effective for HCP removal. Previously proposed mechanisms that contribute to co‐purification of HCPs with the therapeutic protein are either HCP‐drug association or leaching from chromatin heteroaggregates. In this study, we analyzed protein A eluates of 23 Fc‐based proteins by LC‐MS/MS to determine their HCP content. The analysis revealed a high degree of heterogeneity in the number of HCPs identified in the different protein A eluates. Among all identified HCPs, the majority co‐eluted with less than three Fc‐based proteins indicating a drug‐specific co‐purification for most HCPs. Only ten HCPs co‐purified with over 50% of the 23 Fc‐based proteins. A correlation analysis of HCPs identified across multiple protein A eluates revealed their co‐elution as HCP groups. Functional annotation and protein interaction analysis confirmed that some HCP groups are associated with protein‐protein interaction networks. Here, we propose an additional mechanism for HCP co‐elution involving protein‐protein interactions within functional networks. Our findings may help to guide cell line development and to refine downstream purification strategies.