Comprehensive host cell proteins profiling in biopharmaceuticals by a sensitivity enhanced mass spectrometry strategy using TMT-labeling and signal boosting.

Background: Host Cell Proteins (HCPs) are impurities expressed in host cells during the biopharmaceutical production process, whichmay compromise product quality and potentially leading to immunogenic reactions or other adverse effects. Mass spectrometry (MS)-based strategy is more and more considered as a promising method for HCPs analysis, since it is capable of simultaneously quantifying thousands of proteins in a single test. However, considering the large excess biopharmaceutical product protein present in the system and the extremely low abundance of HCPs, sensitive MS methods are urgently needed in HCPs analysis.

Results: In this work, we introduced a novel approach that leveraged host cell lysate as a boosting channel to enhance the MS signal of the residue HCPs in biopharmaceutical products using isobaric TMT labeling, thereby elevating the low-abundant HCPs to detectable and quantifiable levels of current MS without using enrichment or depletion method to avoid disturbance of the original concentration of the HCPs. Our method surpassed previous benchmarks by identifying a significantly higher number (23844 unique peptides for 3475 proteins) compared to existing records (4541 unique peptides for 848 proteins) for HCPs analysis in RM8671 NIST monoclonal antibody (mAb), demonstrating unparalleled sensitivity and robustness. Furthermore, our workflow successfully identified 44 of 48 UPS1 proteins across a concentration range of 0.32-4.15 ppm in monoclonal antibodies (mAbs), proving its effectiveness for in-depth HCPs analysis in biopharmaceuticals.

Significance: Present even at sub-ppm levels, HCPs may compromise the stability and safety of product proteins and alter pharmacokinetics or neutralization of therapeutic effects. Our MS signal enhancing method presented an advancement in HCP analysis, combining improved sensitivity and increased scale of HCPs with a streamlined and robust workflow. This method allowed HCPs quantification at <1 ppm level without disturbance of the original HCPs concentration, which is still rare in the field.