Biosimilar Peptide Mapping Characterization and MAM Workflow Using Prototype Benchtop QTOF with an App-Based Acquisition and Data Processing Platform

Significance of the Topic

Peptide mapping is a cornerstone technique for detailed posttranslational modification (PTM) analysis and comparability assessment of monoclonal antibodies in both innovator and biosimilar biopharmaceutical development. Ensuring the safety, efficacy, and regulatory compliance of these products depends on robust, high-throughput workflows that deliver comprehensive sequence coverage and precise quantitation of critical quality attributes (CQAs).

Study Objectives and Overview

This work aimed to establish a streamlined peptide characterization and Multi-Attribute Method (MAM) workflow for infliximab biosimilars using the prototype Xevo G3 QTof benchtop mass spectrometer integrated with an app-based acquisition and processing platform. Key goals included achieving >95% sequence coverage, comparing PTM profiles between innovator (Remicade) and three biosimilars (Inflectra, Avsola, Renflexis), and evaluating method robustness under thermal stress conditions.

Methodology and Instrumentation

Samples were reduced, alkylated, and digested with trypsin before separation on an ACQUITY Premier UPLC system with reversed-phase chromatography. Data-independent acquisition (DIA, MSE) was performed on the Xevo G3 QTof mass spectrometer. App-based workflows in the UNIFI platform enabled automated data acquisition, processing, and review. Targeted monitoring of PTMs employed the Peptide MAM App.

Results and Discussion

– Sequence coverage exceeded 95% for all four infliximab products, as shown by overlaid chromatograms demonstrating near-identical retention profiles.
– Comparative analysis revealed distinct relative abundances of 13 major N-glycoforms among innovator and biosimilar samples, highlighting subtle biosimilarity differences.
– Key PTMs (oxidation, deamidation, C-terminal Lys heterogeneity) were quantified with the MAM App, exhibiting less than 2% run-to-run RSD.
– A two-week thermal stress study of Remicade and Inflectra demonstrated minor attribute shifts, effectively captured by mirror-plot comparison and quantitative MAM reporting.

Benefits and Practical Applications

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The integrated QTof/app approach reduces noise and file size via intelligent data capture, enabling high-throughput throughput.

Automated workflows improve GMP compliance and minimize manual processing errors.

Robust reproducibility supports trend analysis for biosimilarity assessments and stability studies.

Comprehensive PTM profiling accelerates comparability studies during development and release testing.

Instrumentation Used

• ACQUITY Premier UPLC System
• Xevo G3 QTof Mass Spectrometer
• UNIFI App Peptide Mapping Workflow
• Peptide MAM App for targeted attribute monitoring

Future Trends and Application Prospects

Emerging directions include leveraging artificial intelligence for automated spectral interpretation, expanding MAM workflows to additional biologic classes, and enhancing instrument throughput with continuous-flow ion sources. Integration with cloud-based data platforms may further streamline global QC and regulatory submissions.

Conclusion

The prototype Xevo G3 QTof platform, coupled with app-based acquisition and processing, delivers a robust, high-coverage peptide mapping and MAM workflow. This approach reliably discriminates PTM profiles of innovator and biosimilar infliximab products, supporting efficient biosimilarity assessments in a GMP environment.