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 remains a cornerstone technique for detailed posttranslational modification (PTM) analysis and comparability assessment of therapeutic monoclonal antibodies. Rigorous characterization of innovator versus biosimilar products ensures that safety, efficacy, and regulatory requirements are met throughout biopharmaceutical development.

Objectives and Study Overview

This study aims to implement and validate a peptide mapping and Multi-Attribute Method (MAM) workflow for infliximab biosimilars using a prototype benchtop Xevo™ G3 QTof mass spectrometer. Three biosimilars (Inflectra®, Avsola®, Renflexis®) are compared with the innovator Remicade® to evaluate sequence coverage, PTM profiles, and stress-induced changes, all processed through an app-based acquisition and data analysis platform.

Methodology and Used Instrumentation

Samples of reduced and alkylated infliximab were digested with trypsin and separated by reversed-phase UPLC. Data-independent acquisition (DIA/MSE) was performed on the Xevo™ G3 QTof.

• Chromatography: ACQUITY™ Premier UPLC system
• Mass spectrometry: Xevo™ G3 QTof with enhanced ion optics
• Software: UNIFI™ App Peptide Mapping workflow for attribute characterization and Peptide MAM App for targeted attribute monitoring
• Data platform: waters_connect™ for real-time acquisition, noise reduction, file size optimization, processing, and reporting

Main Results and Discussion

More than 95% sequence coverage was obtained for Remicade® and all three biosimilars. Overlayed chromatograms demonstrated high similarity, while detailed analysis revealed differences in the relative abundance of 13 out of 28 monitored N-glycoforms. Targeted MAM profiling quantified key attributes such as oxidation, deamidation, and C-terminal lysine variants, with consistency across replicates. A two-week thermal stress study of Remicade® versus Inflectra® showed only minor modifications, with method repeatability assessed at ~2% run-to-run relative standard deviation.

Benefits and Practical Applications

The integrated workflow offers:

• Robust, GMP-compliant peptide mapping suitable for biosimilarity studies
• High-throughput data acquisition enabled by real-time noise reduction and file compression
• Comprehensive PTM profiling and quantification to support comparability and regulatory submissions
• App-based automation for streamlined data processing and reporting

Future Trends and Opportunities

Advances in benchtop QTof technology and app-driven informatics are expected to drive further automation and sensitivity improvements. Integration of artificial intelligence for PTM identification, expanded MAM panels for broader attribute coverage, and harmonized regulatory frameworks will enhance the utility of peptide mapping in biosimilar development and quality control.

Conclusion

The demonstrated workflow on the Xevo™ G3 QTof with an app-based platform provides high sequence coverage, precise PTM quantitation, and robust comparability assessment for infliximab biosimilars. Its reproducibility and efficiency support its adoption for routine biosimilarity evaluation and broader biopharmaceutical analytics.