Integrated native digestion and molecular weight cutoff enrichment with DIA-MS: a sensitive and selective approach for quantitative analysis of HCP.

Residual host cell proteins (HCPs) in biopharmaceutical production processes not only compromise drug efficacy but also pose risks to patient safety and product stability, particularly when high-risk HCPs are present. Continuous monitoring of HCPs content and species during downstream purification is therefore critical. Although liquid chromatography-mass spectrometry-based proteomics has emerged as a promising approach for HCP identification, its application is hindered by the substantial dynamic range disparity between high-abundance therapeutic proteins and trace-level HCPs (at parts-per-million, ppm, concentrations). Here, we developed an innovative workflow that eliminates conventional therapeutic protein pre-separation steps for increasing HCP detection. By integrating a two-stage native digestion strategy with molecular weight cutoff filtration, efficient HCP separation and enrichment were achieved. Mass spectrometry data were acquired in data-independent acquisition mode and processed using Spectronaut software for spectral library construction. This integrated approach enabled sensitive detection of HCPs down to 0.5 ppm and continuous quantitative tracking of critical impurities such as the high-risk protein PLBD2, thereby providing real-time monitoring of antibody purification processes and supporting process optimization. Comparative studies with ELISA demonstrated superior sensitivity and specificity of our approach, while systematic method validation confirmed its compliance with bioanalytical requirements, establishing the robustness of the proposed methodology.