Improved identification of host cell proteins in monoclonal antibodies by combining filter-aided sample preparation and native digestion

Abstract

Host cell proteins (HCPs) in biotherapeutics can present potential safety risks or compromise product stability at trace levels. Therefore, removal, testing, and characterization of HCPs are critical throughout biotherapeutic process development. While enzyme-linked immunosorbent assay (ELISA) is the gold standard for quantifying HCPs, it does not provide information on HCP identity. As a result, HCP analysis by liquid chromatography-mass spectrometry (LC-MS) has gained prominence for its ability to identifying HCPs. However, compared to ELISA’s parts-per-billion sensitivity, LC-MS is limited by its dynamic range, often unable to cover magnitude difference between HCP and biotherapeutic concentrations. Thus, an effective HCP enrichment method is highly desirable. In this study, we propose a new strategy for HCP identification that combines filter-aided sample preparation (FASP) with native digestion, followed by shotgun proteomics analysis. This approach improves mAb removal compared to standard native digestion, enabling greater HCP enrichment and identification. Our method detects all spiked proteins at 1 ppm and most at 0.5 ppm, ranging from 12 to 470 kDa, demonstrating broad molecular weight coverage of HCPs. A proof-of-concept analysis using the NISTmAb standard demonstrates that our filter-aided native digestion identifies 155 more HCPs than the standard native digestion. When applying this strategy to an in-house antibody with low amounts of HCPs, we can quantify HCPs at levels as low as 0.03 ppm, demonstrating the high sensitivity in HCP characterization.