High-Risk Host Cell Protein Profiling with Sub-ppm Sensitivity by iRT-Assisted Targeted Mass Spectrometry.

Host cell proteins (HCPs), particularly high-risk species, are critical process-related impurities that can affect the quality, safety, and efficacy of biopharmaceuticals. We developed iRT-assisted targeted mass spectrometry (iRTarget-MS), a robust platform for profiling 31 high-risk HCPs in Chinese Hamster Ovary (CHO) cells across five risk categories: drug aggregation, drug degradation, polysorbate degradation, immunogenic response, and direct biological activity. Optimized for multiplexed, reproducible, and sensitive quantification, iRTarget-MS incorporated iRT values for retention time calibration, enabling the confident identification of low-abundance HCPs. Compared to conventional shotgun proteomics, iRTarget-MS demonstrated significant sensitivity improvement at the subppm level. For example, PLBL2 quantification by iRTarget-MS demonstrated comparable yet more sensitive results compared with the protein-specific enzyme-linked immunosorbent assay (ELISA). Case studies have highlighted the broad applications of iRTarget-MS, including supporting high-throughput process optimization, verification of complete HCP removal while mapping its clearance pathways, and antibody coverage analysis for high-risk HCP subsets. In summary, iRTarget-MS serves as a transformative tool that complements ELISA and shotgun proteomics for high-risk HCP analysis, enhancing the process understanding and accelerating process development. With its ease of operation, streamlined data analysis, and accessible instrumentation, iRTarget-MS opens up opportunities for adopting liquid chromatography-mass spectrometry (LC-MS)-based HCP analysis as a routine monitoring strategy for large sample sets in the biopharmaceutical industry.