Application of a novel LC-MS-based proteomic workflow for host cell protein monitoring and risk control in
Xiaojuan Yu1, Jianhui Cheng2,3, Qianchuan Lv2,3
1State Key Laboratory of Drug Regulatory Science, NHC Key Laboratory of Research on Quality and Standardization of Biotech Products, NMPA Key Laboratory for Quality Research and Evaluation of Biological Products, National Institutes for Food and Drug Control, Beijing 100061, China.
Background:
Comprehensive monitoring of host cell proteins (HCPs) is essential for ensuring the quality, safety, and consistency of antibody therapeutics. Conventional enzyme-linked immunosorbent assay-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 liquid chromatography-mass spectrometry-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 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 nonreducing capillary electrophoresis-sodium dodecyl sulfate purity fluctuations. In the formulation stage, lipoprotein lipase 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 data-independent acquisition (DIA)-mass spectrometry (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.
