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.