Advancing Host Cell Protein Characterization in Monoclonal Antibody Therapeutics: A Nanoparticle-Enriched and High-Sensitivity Mass Spectrometry Approach

Host cell protein (HCP) characterization in monoclonal antibody (mAb) products is challenged by a large concentration gap between low-abundance HCPs and high-abundance mAb proteins. Advances in sample preparation, chromatography, and mass spectrometry techniques have aimed at either reducing this concentration imbalance or the peptide coelution effects during chromatography. Despite this, comprehensive characterization of HCPs remains difficult, particularly at below-ppm levels, often requiring complex and low-throughput methodologies. This study presents a rapid, sensitive, and high-throughput HCP characterization workflow integrating the Seer Proteograph XT nanoparticle-enrichment platform, the Evosep ONE LC system, and timsUltra AIP mass spectrometer. This workflow enables efficient sample preparation (accommodating 40-sample digestions: 20 mAb samples within 6 h) and a 40-sample-per-day LC–MS method. Using a dynamic protein standard, the method achieved a lowest reproducible protein spike-in of 0.2 ppm, outperforming the native digestion approach (∼1 ppm). Application to NISTmAb 8671, identified over 350 HCPs, including those at sub-ppm levels, confidently and reproducibly. Furthermore, analysis of three commercial products identified 80–200 additional HCPs undetected by native digestion. Overall, our results highlight the potential of automated nanoparticle enrichment and advanced mass spectrometry for high-throughput, sensitive, and reproducible detection of low-level HCPs, enabling rapid risk assessment and improved biopharmaceutical purification strategies.