Characterization of IgG1 monoclonal antibody (mAb) oxidation variants at intact, subunit and peptide levels

Significance of the Topic

Accurate characterization of monoclonal antibody oxidation variants is critical to ensure biotherapeutic stability, safety and efficacy.
Oxidation can alter pharmacokinetics, immunogenicity and potency, making its detection and localization essential during development and quality control.

Objectives and Overview

This study aimed to demonstrate the capability of the Orbitrap Exploris 240 mass spectrometer for site-specific identification of methionine oxidation in the IgG1 antibody ipilimumab.
Analyses were conducted at intact protein, subunit and peptide levels under controlled oxidative stress conditions.

Methodology and Instrumentation

Samples of ipilimumab were treated with hydrogen peroxide (50–500 ppm H2O2) for 24 hours.

• Intact protein and subunits were analyzed by LC-MS using a Vanquish Duo UHPLC system coupled to an Orbitrap Exploris 240 MS.
• Proteolytic subunits were generated by IdeS digestion followed by reduction; peptides by SMART Digest trypsin kits on KingFisher Duo Prime.
• Columns: MAbPac RP for intact and subunit, Acclaim C18 for peptide mapping.
• Data were processed using Xcalibur and BioPharma Finder software.

Main Results and Discussion

Intact Mass Analysis:
Oxidative stress induced a +64 Da shift, consistent with four methionine oxidations; spectral clarity allowed baseline glycoform resolution.

Subunit Analysis:
Fc/2 subunit exhibited one and two oxidation events detected at +16 and +32 Da shifts with accurate deconvolution of glycoforms.

Peptide Level Mapping:
The tryptic peptide DTLMISR showed a +15.9949 Da mass increase and earlier elution upon oxidation.
MS/MS fragmentation provided retention of y4 and y5 ions with diagnostic neutral loss of CH3SOH, confirming methionine oxidation sites.

Benefits and Practical Applications of the Method

• Site-specific identification of oxidation hotspots enhances CQA monitoring in biopharma pipelines.
• The workflow demonstrates versatility across analysis levels with minimal sample preparation.
• High resolution and mass accuracy enable detection of low-level modifications with confidence.

Future Trends and Opportunities

Emerging high-resolution accurate-mass instruments will further improve throughput and sensitivity for PTM characterization.
Integration with automated digestion and AI-driven data analysis may enable real-time monitoring during manufacturing.

Conclusion

The Orbitrap Exploris 240 platform combined with streamlined LC-MS methods provides robust, high-confidence characterization of mAb oxidation variants, supporting quality assurance and process development in biopharmaceutical research.

References

• Carillo S. and Bones J., NIBRT, Dublin, Ireland; Buchanan T., Srzentic K. and Scheffler K., Thermo Fisher Scientific. Application Brief 73555.