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.

宿主细胞蛋白（Host cell proteins, HCPs），尤其是高危亚型，是一类关键的工艺相关杂质，可影响生物制药产品的质量、安全性与有效性。本研究开发了iRT辅助靶向质谱法（iRT-assisted targeted mass spectrometry, iRTarget-MS），这是一款可用于分析中国仓鼠卵巢（Chinese Hamster Ovary, CHO）细胞中31种高危HCP的稳健平台，覆盖5类风险范畴：药物聚集、药物降解、聚山梨酯降解、免疫原性反应及直接生物活性。

该平台针对多重检测、可重现性及灵敏定量进行了优化，引入iRT值用于保留时间校准，可实现低丰度HCP的可靠鉴定。相较于传统鸟枪法蛋白质组学，iRTarget-MS在亚ppm级别展现出显著的灵敏度提升。例如，针对PLBL2的定量分析显示，iRTarget-MS的结果与蛋白特异性酶联免疫吸附试验（enzyme-linked immunosorbent assay, ELISA）相当，但灵敏度更优。

案例研究凸显了iRTarget-MS的广泛应用场景，包括支撑高通量工艺优化、验证HCP的完全清除并绘制其清除通路、以及针对高危HCP亚群的抗体覆盖分析。

综上，iRTarget-MS是一款变革性分析工具，可作为ELISA与传统鸟枪法蛋白质组学的补充手段用于高危HCP分析，强化工艺认知并加速工艺开发进程。凭借操作简便、数据分析流程化及仪器设备可及性强的优势，iRTarget-MS为液相色谱-质谱联用法（liquid chromatography–mass spectrometry, LC-MS）驱动的HCP分析成为生物制药行业大样本集的常规监测策略提供了可行路径。