Application of a Novel LC-MS-Based Proteomic Workflow for Host Cell Protein Monitoring and Risk Control in Biotherapeutics

Abstract Background Comprehensive monitoring of host cell proteins (HCPs) is essential for ensuring the quality, safety, and consistency of antibody therapeutics. Conventional ELISA-based assays provide only total HCP burden and lack the resolution to identify individual, functionally active impurities that may affect product stability or efficacy. To address this limitation, we developed a versatile, high-sensitivity mass spectrometry–based workflow capable of molecular-level HCP characterization and risk assessment. Methods We developed and evaluated a sub-ppm, easy-to-use LC-MS-based proteomics workflow for the identification and relative quantitation of HCPs across downstream purification and final formulation, which offers molecular-specific, risk-based process characterization and regulatory-aligned quality assurance for antibody therapeutics. Results The workflow demonstrated sub-ppm sensitivity and excellent reproducibility, enabling comprehensive tracking of HCP clearance across the purification process. Scale-up from 100 L to 500 L led to higher total HCP levels and moderately increased species diversity, indicating reduced clearance efficiency and altered impurity profiles. Mechanistic interpretation linked transient thioredoxin and thioredoxin reductase presence to disulfide bond reduction and nrCE–SDS purity fluctuations. In the formulation stage, lipoprotein lipase (LPL) was identified as a key contributor to polysorbate 80 (PS80) degradation, consistent with accelerated stability results, leading to successful process optimization. Conclusions This study establishes a comprehensive, sub-ppm DIA-MS platform for HCP monitoring in antibody therapeutics, bridging analytical characterization with process and formulation control. The workflow enables molecular-level understanding of impurity behavior, supports proactive risk mitigation, and enhances overall process consistency, representing a significant advancement in HCP management and biotherapeutic quality assurance.