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

Area of Science:

• Biopharmaceutical analysis
• Proteomics
• Analytical chemistry

Background:

• Host cell proteins (HCPs) are critical process-related impurities in biopharmaceuticals, impacting product quality and patient safety.
• Targeted LC-MS/MS assays like multiple reaction monitoring (MRM) are used for HCP quantification but struggle with retention time (RT) variability in complex matrices.
• This variability limits the transferability and multiplexing capabilities of conventional MRM workflows.

Purpose of the Study:

• To address the limitations of conventional MRM workflows in quantifying host cell proteins (HCPs) across diverse biopharmaceutical matrices.
• To develop a more robust and transferable method for highly multiplexed HCP analysis.
• To improve the accuracy and sensitivity of HCP quantification in biopharmaceutical products.

Main Methods:

• Implementation of a scout-triggered MRM (st-MRM) acquisition strategy based on event-driven monitoring.
• Utilized a targeted assay for 240 peptides from 97 Chinese Hamster Ovary (CHO)-derived HCPs.
• Evaluated method performance across heterogeneous drug substances and process intermediates, assessing peptide detection and signal integrity.

Main Results:

• The st-MRM approach demonstrated robust peptide detection across diverse matrices in a single injection, eliminating the need for method re-optimization.
• Significant improvements in chromatographic peak integrity and peptide signal retention were observed compared to conventional MRM.
• Absolute quantification using stable isotope-labeled peptides achieved a dynamic range of six orders of magnitude, with HCPs detected down to 2.9 ppm in purified drug substances.

Conclusions:

• Scout-triggered MRM (st-MRM) significantly enhances the robustness and transferability of highly multiplexed targeted proteomics workflows for HCP analysis.
• The st-MRM strategy provides a reliable solution for overcoming RT variability challenges in complex biopharmaceutical samples.
• This advancement facilitates more accurate and sensitive monitoring of HCP impurities, contributing to improved biopharmaceutical quality and patient safety.