Host cell protein networks as a novel co-elution mechanism during protein A chromatography,Biotechnology and Bioengineering

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Host cell protein networks as a novel co-elution mechanism during protein A chromatography
Biotechnology and Bioengineering ( IF 3.8 ) Pub Date : 2024-03-07 , DOI: 10.1002/bit.28678
Sherin Panikulam _{1,

2} , Alexander Hanke ₃ , Frieder Kroener ₃ , Anette Karle ₄ , Oliver Anderka ₃ , Thomas K. Villiger ₁ , Nicolas Lebesgue ₃

Affiliation

Host 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.

更新日期：2024-03-07

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