Divergent host cell protein profiles during special purification of biologics from prokaryotic versus eukaryotic systems dictate the need for tailored ELISA assay development.

Therapeutic antibodies are expressed via host systems, such as Chinese hamster ovary (CHO) cells and Escherichia coli (strain B / BL21-DE3, E.coli BL21), followed by purification to remove host cell proteins (HCPs), thereby averting risks including immunogenicity, antibody degradation, and protein aggregation. During regulatory filing, drug-specific HCP ELISA kits custom-developed using null-transfected harvest cell culture fluid (HCCF) are typically required to ensure accurate HCP quantification. In a case involving an E.coli-expressed antibody, initial HCP ELISA kits derived solely from HCCF significantly underestimated HCP levels in the purification intermediate (cation-exchange chromatography fraction, CEX1). Supplementing anti-CEX1 HCP antibodies significantly improved detection capability. Subsequent high-resolution mass spectrometry (HRMS) characterization revealed substantial shifts in HCP composition and abundance between HCCF and CEX1. Conversely, for CHO cell-derived antibodies (eukaryotic system), no significant alteration in HCP profiles was observed between HCCF and the analogous purification intermediate. This divergence provides critical insights for optimizing HCP analytical strategies of special purification antibody therapeutics.