Deep dive into drug substance HCP patterns for comprehensive safety assessment in recombinant mAb formats.

Abstract

The presence of host cell proteins (HCPs) as process-related impurities in monoclonal antibody (mAb) therapeutics presents risks, including immunogenicity, product instability, and reduced therapeutic efficacy. Therefore, HCP levels are seen as a critical quality attribute which requires monitoring. Characterization of residual HCPs is essential during process development and after process changes in late project phases including post-submission changes. Mass spectrometry-based analysis of HCPs is increasingly employed to overcome the limitations of ELISAs, particularly in identifying individual low-abundance HCPs. In this study, we systematically compared three enrichment strategies: hexapeptide enrichment, immunoaffinity chromatography (IAC), and native digest (ND), to enhance LC-MS/MS-based detection of low-abundance HCPs across five recombinant products expressed in CHO cells. Our results show that both ND and IAC substantially expand HCP detection beyond hexapeptide enrichment and in-solution digest in total number of HCP identification. While IAC supported the detection of several potentially critical HCPs, ND uniquely identified additional low-abundance HCPs not captured by antibody-based methods. Furthermore, pull-down assays revealed product-specific HCPs likely interacting with the active pharmaceutical ingredient, suggesting that standard purification steps may be insufficient for their removal. Together, our findings emphasize the complementary nature of ND and IAC for in-depth HCP profiling. We propose the combined application of both methods to ensure comprehensive detection of potentially critical HCPs in biopharmaceutical products, particularly when facing issues with low product or polysorbate stability caused by HCPs with enzymatic activities. This integrative approach supports enhanced product safety and control of consistent HCP depletion in the process.