Peptide Mapping and Quantitation of Oxidation and Deamidation in Monoclonal Antibodies

Importance of the Topic

Monoclonal antibodies (mAbs) represent a major class of biopharmaceuticals, offering targeted therapeutic effects. Ensuring the correct primary structure and monitoring post-translational modifications (PTMs) such as oxidation and deamidation is critical for product efficacy and safety. High-resolution peptide mapping by LC/MS combined with automated sample preparation enhances reliability and throughput in quality control of mAbs.

Objectives and Study Overview

This application note presents a fully integrated workflow for verifying mAb identity and quantifying chemically induced oxidation and deamidation. Two samples—NISTmAb reference material and rituximab—were evaluated to demonstrate sequence confirmation and site-specific PTM quantitation under defined stress conditions.

Methodology

Automated digestion was performed on the Agilent AssayMAP Bravo platform to reduce human error and improve reproducibility. Oxidation stress was applied using hydrogen peroxide at 0–0.2% (v/v) for 24 hours at room temperature. Deamidation was induced by incubating samples in Tris buffer (pH 8.9) at 37 °C for three days. After C18 cleanup, peptides were separated on an Agilent 1290 Infinity II Bio LC with an AdvanceBio Peptide Mapping column and detected using the Agilent 6545XT AdvanceBio LC/Q-TOF. Data processing and PTM localization were carried out in Agilent MassHunter BioConfirm software.

Used Instrumentation

• Agilent AssayMAP Bravo automated sample prep system
• Agilent 1290 Infinity II Bio LC with AdvanceBio Peptide Mapping column
• Agilent 6545XT AdvanceBio LC/Q-TOF with Dual Jet Stream ESI source
• Agilent MassHunter BioConfirm software version 12.1

Key Results and Discussion

• Sequence coverage for both NISTmAb and rituximab exceeded 95%, confirming primary structure integrity.
• Oxidation at M255 (heavy-chain Fc), M4 (light-chain N-terminus), and M256 (rituximab) increased proportionally with H2O2 concentration, reaching near-complete conversion at 0.2% H2O2. Quantitation demonstrated excellent reproducibility (SD < 2.5%, n=4).
• Deamidation of the peptide NQVSLTCLVK yielded distinct chromatographic peaks for isoforms. MS/MS fragment shifts enabled confident assignment of modifications at Q365 and N364, even at low abundance.

Benefits and Practical Applications

The automated workflow minimizes variability and contamination risk while accelerating sample throughput. Combined with high-resolution LC/Q-TOF detection and advanced data analysis in BioConfirm, it provides a robust platform for routine PTM monitoring, essential for mAb development, stability testing, and regulatory compliance.

Future Trends and Opportunities

• Adoption of higher-throughput automation and multiplexed sample processing.
• Integration of machine learning for automated PTM detection and pattern recognition.
• Expansion of the workflow to characterize additional PTMs and complex biologics beyond mAbs.

Conclusion

The Agilent-based integrated workflow combining automated digestion, high-resolution peptide mapping, and specialized software delivers accurate, reproducible mapping and quantitation of oxidation and deamidation in monoclonal antibodies. This approach strengthens analytical rigor and efficiency in biopharmaceutical quality control.

References

1. ICH Q6B: Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products. ICH Guideline, 1999.
2. Gupta S, Jiskoot W, Schöneich C, Rathore AS. Oxidation and Deamidation of Monoclonal Antibody Products: Potential Impact on Stability, Biological Activity, and Efficacy. J Pharm Sci. 2022;111(4):903–918.
3. Bovee M, Russell J, Murphy S. Automation of Sample Preparation for Accurate and Scalable Quantification and Characterization of Biotherapeutic Proteins Using the Agilent AssayMAP Bravo Platform. Agilent Technologies Application Note 5991-4872EN, 2016.
4. Haberger M et al. Assessment of Chemical Modifications of Sites in the CDRs of Recombinant Antibodies. mAbs. 2014;6(2):327–339.