Assessing Oxidation in IgG1 Monoclonal Antibodies and Correlating at both Intact Protein and Peptide Levels

Significance of the Topic

Oxidation of methionine residues represents a critical quality attribute in therapeutic monoclonal antibodies, directly affecting product safety, efficacy and stability.

Objectives and Study Overview

This study assessed the performance of the Thermo Scientific Orbitrap Exploris 240 mass spectrometer equipped with the BioPharma option for comprehensive oxidation analysis. Two IgG1 antibodies, ipilimumab and golimumab, were incubated with hydrogen peroxide (50, 100 and 500 ppm) for 24 hours to induce oxidative stress. Analyses were performed at the intact protein, subunit and peptide levels to pinpoint oxidation hotspots and validate orthogonal characterization.

Applied Methodology and Instrumentation

Reversed-phase liquid chromatography coupled to high-resolution mass spectrometry was used across three workflows:

• Intact mass analysis under denaturing conditions (RP LC-MS) using a Vanquish Duo UHPLC and MAbPac RP column at 80 °C.
• Subunit analysis following IdeS proteolysis, reduction, and RP LC-MS separation of Fc/2, light chain and Fd′ fragments.
• Peptide mapping via SMART Digest trypsin kits on a KingFisher Duo Prime system, separation on an Acclaim Vanquish C18 column at 25 °C, and MS/MS with HCD fragmentation.

Data processing and deconvolution were performed with BioPharma Finder 4.0. The Exploris 240 was operated with templated acquisition settings, achieving resolutions up to 120,000 and mass accuracy <1 ppm.

Main Results and Discussion

At the intact level, treatment with 500 ppm H2O2 produced a mass shift of ~64 Da, indicating oxidation of four methionine residues and resolving glycoform patterns after deconvolution. Subunit analysis revealed dose-dependent increases in oxidation at specific methionine sites within Fc and Fd′ domains, quantified as % abundance. Peptide mapping highlighted the heavy-chain peptide DTLMISR with a +15.9949 Da mass increase and earlier chromatographic elution (RT 16.04 vs. 18.47 min). MS/MS spectra confirmed oxidation by characteristic y4 and y5 fragments and diagnostic loss of CH3SOH.

Benefits and Practical Applications

The Orbitrap Exploris 240 platform delivers high-throughput, accurate tracking of post-translational modifications with simplified spectral interpretation and templated method transfer. Combining intact, subunit and peptide data provides robust, orthogonal confirmation of oxidation sites, supporting quality control in biopharmaceutical development and manufacturing.

Future Trends and Applications

Integration of automated data workflows and real-time monitoring could further accelerate PTM profiling. Expansion to other biotherapeutic formats, such as fusion proteins and antibody–drug conjugates, will broaden the utility of multi-level MS characterization under diverse stress conditions.

Conclusion

The Thermo Scientific Orbitrap Exploris 240 with BioPharma option enables sensitive, accurate and operationally simple assessment of methionine oxidation in monoclonal antibodies across structural hierarchies, enhancing confidence in critical quality attribute characterization.

Applied Instrumentation

• Thermo Scientific Vanquish Duo UHPLC
• Orbitrap Exploris 240 Mass Spectrometer with BioPharma option
• KingFisher Duo Prime Purification System
• SMART Digest Trypsin kits
• BioPharma Finder 4.0 software

References

1. Kuo T et al, mAbs 2011, 3, 422–430
2. Guan Z et al, J Am Soc Mass Spectrom 2003, 14, 605–613
3. Pan H et al, Protein Sci. 2009, 18, 424–433