Confident peptide mapping and disulfide bond analysis of an IgG2 monoclonal antibody

Importance of the Topic

The detailed characterization of monoclonal antibodies is essential in biopharmaceutical development to ensure product safety, efficacy and consistency. Peptide mapping combined with disulfide bond analysis provides insights into the primary structure and higher-order arrangement of antibodies, enabling monitoring of critical quality attributes (CQAs) throughout manufacturing and regulatory approval.

Study Objectives and Overview

This work demonstrates the application of the Thermo Scientific Orbitrap Exploris 240 mass spectrometer for routine LC-MS peptide mapping of an IgG2 monoclonal antibody (denosumab). Goals include achieving full amino acid sequence coverage, confident identification of inter- and intrachain disulfide bonds, and sensitive detection of low-level post-translational modifications (PTMs) under both reducing and non-reducing conditions.

Methodology and Instrumentation

Sample preparation was performed using automated proteolytic digestion under reducing and non-reducing conditions on a KingFisher Duo Prime system with SMART Digest Trypsin kits. Liquid chromatography employed a Vanquish Duo UHPLC system with a C18 column (2.1×250 mm, 2.2 μm) and a water/acetonitrile gradient with 0.1% formic acid. Mass spectrometry analysis was conducted on an Orbitrap Exploris 240 mass spectrometer with BioPharma Option.

• MS1 resolution: 120 000 FWHM at m/z 200
• MS2 resolution: 15 000 FWHM at m/z 200, data-dependent top 5 acquisition
• Data processing: Xcalibur 4.2 and BioPharma Finder 4.0

Key Results and Discussion

The peptide mapping assay delivered 100 % sequence coverage for both light and heavy chains of denosumab, including complementarity-determining regions (CDRs). Disulfide bond mapping under non-reducing conditions identified all expected intra- and interchain linkages with precursor and fragment mass accuracy below 3 ppm. A low-abundance deamidation at N362 was detected at approximately 2.5 % relative abundance with clear chromatographic separation and high-resolution MS/MS validation.

Benefits and Practical Applications

• Complete confirmation of antibody primary structure and disulfide bond arrangement.
• High-confidence assignment of low-level PTMs supporting quality control.
• Operational simplicity and fast method transferability across laboratories.

Future Trends and Applications

Advancements in high-resolution mass spectrometry are expected to further streamline multi-attribute methods, increase throughput and enhance automation. Integration with machine learning for data interpretation and real-time monitoring of CQAs will drive next-generation biopharmaceutical analytics.

Conclusion

The Orbitrap Exploris 240 mass spectrometer enables reliable peptide mapping, disulfide bond analysis and sensitive PTM detection for monoclonal antibodies. Its ease of use, robust performance and transferability make it ideal for routine CQA monitoring in biopharmaceutical development and production.