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

Area of Science:

• Biopharmaceutical Analysis
• Proteomics
• Analytical Chemistry

Background:

• Host cell proteins (HCPs) are critical impurities in biopharmaceuticals, impacting product quality and safety.
• High-risk HCPs pose significant threats, including drug aggregation, degradation, and immunogenic responses.
• Accurate and sensitive detection of HCPs is essential for biopharmaceutical development and quality control.

Purpose of the Study:

• To develop and validate a robust platform, iRT-assisted targeted mass spectrometry (iRTarget-MS), for profiling high-risk HCPs.
• To enhance the sensitivity and reproducibility of HCP quantification in Chinese Hamster Ovary (CHO) cells.
• To demonstrate the broad applicability of iRTarget-MS in biopharmaceutical process development.

Main Methods:

• Development of iRT-assisted targeted mass spectrometry (iRTarget-MS) for profiling 31 high-risk HCPs.
• Optimization for multiplexed, reproducible, and sensitive quantification using iRT values for retention time calibration.
• Comparison with conventional shotgun proteomics and enzyme-linked immunosorbent assay (ELISA) for sensitivity and accuracy.

Main Results:

• iRTarget-MS demonstrated significant sensitivity improvement at the sub-ppm level compared to shotgun proteomics.
• Quantification of PLBL2 by iRTarget-MS showed comparable yet more sensitive results than ELISA.
• Case studies confirmed iRTarget-MS's utility in process optimization, HCP removal verification, and clearance pathway mapping.

Conclusions:

• iRTarget-MS is a transformative tool complementing ELISA and shotgun proteomics for high-risk HCP analysis.
• The platform enhances process understanding and accelerates biopharmaceutical development.
• iRTarget-MS facilitates the routine monitoring of large sample sets using LC-MS-based analysis.