Development, validation, and implementation of a robust and quality control-friendly focused peptide mapping method for monitoring oxidation of co-formulated monoclonal antibodies.

Monoclonal antibody (mAb) coformulation containing two therapeutic proteins provides benefits of improved therapeutic efficacy and better patient compliance. Monitoring of the individual mAb stability in the coformulation is critical to ensure its quality and safety. Among post-translational modifications (PTMs), oxidation is often considered as one of the critical quality attributes (CQAs) as it potentially affects the structure and potency. Although hydrophobic interaction chromatography (HIC) and reversed phase liquid chromatography (RPLC) have been used to monitor overall protein oxidation, mass spectrometry of peptide digests resolved by LC methods can afford superior selectivity and sensitivity for specific PTMs. With the advent of the Quadrupole Dalton (QDa) mass spectrometer as an affordable add-on detector, implementation of targeted oxidation assays in development and quality control (QC) laboratories is now feasible. In this study, as the first effort to implement MS-based methods for antibody coformulation in QC laboratories, we developed and validated a high-throughput and robust focused peptide mapping method using QDa for simultaneous site-specific monitoring of oxidation of methionine and tryptophan residues in heavy-chain (HC) complementary determining regions (CDRs) of two co-formulated mAbs. The method was validated in terms of accuracy, precision, linearity, range, quantitation limit (QL), specificity, and solution stability per recommendations in ICH Q2. The method robustness was systematically assessed involving multiple sample preparation and instrument method parameters. The method met the validation criteria in GMP laboratories with excellent robustness and was implemented in both GMP and development environments.