Host cell proteins (HCPs) are critical process-related impurities in monoclonal antibody (mAb) production that can compromise product safety, efficacy, and stability. Traditional enzyme-linked immunosorbent assays (ELISA) provide total HCP quantification but lack the specificity to detect individual high-risk species. While mass spectrometry (MS) has emerged as a powerful orthogonal approach, offering molecular-level resolution, high sensitivity, and the ability to identify and quantify low-abundance, potentially immunogenic HCPs. This review provides a strategy-oriented and practice-driven analysis of MS-based HCP analytics, emphasizing how recent methodological advances address real-world challenges in bioprocess development and regulatory compliance. We critically examine optimized sample preparation workflows, including buffer and matrix mitigation, native and denaturing digestion, antibody depletion, and HCP enrichment, alongside multidimensional chromatographic separations and advanced acquisition strategies spanning DDA, DIA, and targeted MS. Particular attention is given to emerging hybrid and integrated workflows, such as DDA-PRM-dMRM acquisition schemes, that bridge discovery and routine quality control. Beyond these, our review places MS-based HCP analysis in evolving global regulatory expectations, providing a comparative and practical interpretation of guidance from the USP, EP, JP, BP, and ChP, with emphasis on validation, standardization, and interlaboratory reproducibility. By integrating technical advances with regulatory frameworks and case-based evidence, our review highlights how MS is transitioning from a supportive characterization tool to a core component of risk-based and multiattribute HCP control strategies in modern biopharmaceutical development.
Cao et al. (Tue,) studied this question.