Applying Peptide Mapping and Multi- Attribute Method (MAM) Workflow for Biosimilar mAb Drug Products Comparison on the Xevo™ G3 QTof Platform

Significance of the Topic

Peptide mapping combined with the multi-attribute method (MAM) offers a streamlined and high-throughput approach for the detailed characterization of biosimilar monoclonal antibodies (mAbs). As patents expire and biosimilar products enter a competitive market, robust analytical workflows are essential to ensure that these follow-on therapeutics match the innovator in terms of safety, efficacy, and stability.

Objectives and Study Overview

This study evaluated the performance of the Xevo G3 QTof platform integrated with the compliance-ready waters_connect informatics ecosystem. Four infliximab products (innovator Remicade and three biosimilars: Inflectra, Avsola, Renflexis) were compared using peptide mapping and targeted attribute monitoring. A thermal stress study on Remicade and Inflectra further probed critical quality attributes (CQAs).

Instrumentation

• ACQUITY Premier UPLC System with CSH C18 column at 60 °C
• Xevo G3 QTof mass spectrometer (ESI+; 100–2000 m/z; low/high energy MSE)
• waters_connect informatics platform with UNIFI Peptide Mapping App and Peptide MAM App

Methodology

Infliximab samples were reduced, alkylated, desalted, tryptically digested, and analyzed via reversed-phase LC–MSE. Data independent acquisition enabled precise peptide identification and localization of modifications. System suitability used a MassPREP peptide standard and monitored mass accuracy, peak width, and reproducibility throughout the sequence.

Key Results and Discussion

Sequence coverage exceeded 95% for all four mAbs with mass errors below 5 ppm and injection-to-injection RSDs under 5%. Forty-seven modifications were localized, including deamidation, oxidation, N-glycosylation, and C-terminal lysine clipping. Quantitative differences among products were detected:

• Oxidation and deamidation sites varied in relative abundance across biosimilars versus innovator
• Glycoform profiles (FA2, FA2G1, FA2G2 and immunogenic variants) differed according to cell-line origin
• Lysine clipping percentages revealed production-specific patterns

A two-week thermal stress experiment highlighted shifts in CQAs, with some oxidation sites increasing or decreasing differentially between Remicade and Inflectra, while deamidation trends were parallel.

Benefits and Practical Applications

• High-coverage, reproducible peptide mapping suitable for regulatory comparability
• Integrated, app-based workflow accelerates data acquisition, processing, and review under compliance requirements
• MAM enables simultaneous monitoring of multiple attributes within a single LC–MS run, reducing time and resource demands

Future Trends and Opportunities

Advances are expected in AI-driven data analysis, real-time attribute monitoring, expanded spectral libraries, and standardized MAM protocols across platforms. Integration with automated sample handling and cloud-based compliance could further accelerate biosimilar development and quality control.

Conclusion

The Xevo G3 QTof platform, combined with waters_connect informatics and MAM workflows, delivers a robust solution for comparative peptide mapping of biosimilar mAbs. This integrated approach achieves high sequence coverage, precise quantification of post-translational modifications, and efficient identification of CQAs under stress, supporting both research and regulatory requirements.

References

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