Scout-Triggered Multiple Reaction Monitoring Enables Robust Quantification of Host Cell Proteins Across Bioprocess...

Host cell proteins (HCPs) are process-related impurities that must be monitored in biopharmaceutical products due to their potential impact on product quality and patient safety. Targeted LC-MS/MS approaches such as multiple reaction monitoring (MRM) enable protein-specific HCP quantification but are difficult to apply in highly multiplexed assays because of retention time (RT) variability across complex bioprocess matrices.

Here, we show that conventional RT-scheduled MRM workflows lack transferability when applied to heterogeneous drug substances and process intermediates. Using a targeted assay comprising 240 peptides corresponding to 97 CHO-derived HCPs, RT shifts of several minutes resulted in truncated chromatographic peaks and peptide signal loss, even when wide scheduling windows were used. To overcome this limitation, a scout-triggered MRM (st-MRM) acquisition strategy based on event-driven monitoring was implemented.

This approach enabled robust peptide detection across diverse matrices within a single injection, without method re-optimization. Absolute quantification using stable isotope-labeled peptides spanned six orders of magnitude, with HCPs quantified down to 2.9 ppm in purified drug substances.

Overall, st-MRM improves the robustness and transferability of highly multiplexed targeted proteomics workflows for HCP analysis.