Quantitation of Chemical-Induced Deamidation and Oxidation on Monoclonal Antibodies

Significance of the Topic

This study addresses critical chemical modifications in monoclonal antibodies, namely asparagine deamidation and methionine oxidation, which can compromise protein stability, efficacy, and safety. Monitoring these modifications during formulation, storage, and stress testing is essential for ensuring consistent product quality and therapeutic performance.

Aims and Study Overview

The application note presents an integrated workflow for simultaneous identification and quantification of chemically induced deamidation and oxidation in recombinant monoclonal antibodies. The approach combines automated sample handling, liquid chromatography high resolution mass spectrometry, and specialized data analysis software to map peptide modifications and quantify modification levels across stress conditions.

Methodology and Instrumentation

The workflow consists of automated reduction, alkylation, tryptic digestion, and desalting using the Agilent AssayMAP Bravo platform followed by peptide separation on an Agilent 1290 Infinity II LC system with a reversed-phase C18 column. Detection and data acquisition are performed on the Agilent 6545XT AdvanceBio LC/Q-TOF with a Jet Stream ESI source. Data processing, peptide spectrum matching, and post-translational modification quantitation are carried out in Agilent MassHunter BioConfirm 10.0 software.

• Agilent AssayMAP Bravo automated sample preparation
• Agilent 1290 Infinity II LC with C18 charged-surface column
• Agilent 6545XT AdvanceBio LC/Q-TOF mass spectrometer
• Agilent MassHunter BioConfirm 10.0

Main Results and Discussion

Chromatographic separation resolved seven distinct forms of a model peptide containing three asparagine sites, enabling confident assignment of deamidation at N389 and N394. MS/MS spectral comparison confirmed site localization with y-ion shifts corresponding to +1 and +2 dalton mass changes. Quantitative profiling under high-pH stress revealed N389 deamidation exceeding 80 percent by day 13, while combined N394/N395 modification remained below 20 percent. Oxidation studies using hydrogen peroxide showed a dose-dependent increase in light chain methionine oxidation, demonstrating method sensitivity to oxidative stress conditions.

Benefits and Practical Applications of the Method

The integrated platform accelerates peptide mapping and modification analysis by automating sample prep, delivering high-resolution separation, and enabling streamlined software-driven quantitation. It supports stability studies, forced degradation experiments, and critical quality attribute monitoring in biopharmaceutical development and quality control workflows.

Future Trends and Applications

Advances in automation and data analytics are expected to expand the range of detectable modifications and increase throughput. Integration of machine learning for automated feature detection, real-time feedback loops for process control, and multiplexed assays for simultaneous monitoring of multiple quality attributes will further accelerate therapeutic protein characterization.

Conclusion

The demonstrated workflow provides a robust and comprehensive solution for characterization and quantification of deamidation and oxidation in monoclonal antibodies. By combining automated sample preparation, high-resolution LC/Q-TOF analysis, and dedicated software, the platform ensures accurate monitoring of key modifications critical to biotherapeutic quality and stability.

Reference

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