Platform‐Specific Immunoassay Development and Orthogonal Characterization for Improved Host Cell Protein Control in Vero Cell‐Derived Vaccines

Abstract

Vero host cell proteins (HCPs) are critical process‐related impurities in Vero cell‐derived vaccines and require robust analytical control to support product quality, safety, and manufacturing consistency. During development of the V590 vaccine candidate, discontinuation of the original commercial Vero HCP ELISA prompted evaluation of alternative approaches for HCP monitoring and risk control. We compared first‐ and second‐generation commercial ELISA kits, assessed the impact of process‐representative HCP standards and anti‐HCP antibodies on HCP quantitation, and characterized antibody coverage by two‐dimensional differential‐in‐blot electrophoresis. Measured HCP levels in process intermediates and bulk drug substances were strongly influenced by sample composition, HCP standard, antibody reagents, and assay format. ELISA and Simple Western yielded different quantitative results even with the same standard and antibodies, underscoring the assay‐dependent nature of HCP measurement. Platform‐specific anti‐Vero HCP antibodies provided broader coverage of process‐representative HCPs than the second‐generation commercial ELISA antibodies. Orthogonal characterization by Simple Western and Liquid chromatography‐mass spectrometry (LC‐MS/MS) further enabled monitoring of fibronectin, a high‐risk Vero HCP that causes filter fouling and compromises downstream process performance. Together, these findings demonstrate that platform‐specific immunoassays combined with orthogonal characterization of process‐relevant high‐risk HCPs provide a robust analytical framework for Vero HCP control and long‐term assay lifecycle management in Vero cell‐derived vaccine manufacturing.