A Robust Workflow for Biosimilar Comparability Assessment via Intact and Subunit RPLC-MS and Native IEX-MS with a Prototype Benchtop QTOF

Importance of the Topic

The comparability assessment of biosimilar monoclonal antibodies is critical to ensure safety, efficacy, and regulatory compliance. Advanced liquid chromatography–mass spectrometry techniques provide detailed characterization of charge variants and glycoforms, enabling detection of low-abundance modifications that can affect therapeutic performance.

Objectives and Study Overview

This study aimed to establish a robust analytical workflow on a benchtop quadrupole time-of-flight (QToF) mass spectrometer for intact and subunit-level reversed-phase LC-MS and native ion-exchange MS. It focused on comparing innovator infliximab (Remicade®) with three biosimilars (Inflectra®, Avsola®, Renflexis®) to quantify glycoform distributions and charge variants.

Methodology

• Sample set: Remicade and three biosimilars analyzed in triplicate.
• Intact analysis: Denaturing reversed-phase LC-MS (RPLC) and native ion-exchange (IEX) coupled to QToF.
• Subunit analysis: IdeS digestion to generate Fc and Fd+LC fragments, followed by RPLC-MS and IEX-MS.
• Data processing: UNIFI and waters_connect platforms using MaxEnt1 deconvolution.

Instrumentation Used

• Xevo G3 QTof mass spectrometer (Waters Corporation).
• ACQUITY Premier I-Class UPLC system with Premier BEH C4 column.
• BioResolve SCX mAb column for native IEX separation.
• Inline UV detection at 280 nm.
• UNIFI software and waters_connect informatics for acquisition and review.

Main Results and Discussion

• Intact RPLC-MS mirror plots revealed distinct C-terminal lysine variant profiles and N-glycoform compositions among biosimilars relative to Remicade, with confident detection of variants down to <5% abundance.
• Native IEX-MS confirmed three major charge variant peaks corresponding to C-terminal lysine isoforms; carboxypeptidase B digestion validated lysine assignments.
• Subunit analysis enabled quantitation of Fc glycoforms to 1% level, detecting immunogenic glycans (NeuGc, alpha-Gal) in Remicade and Inflectra.
• Relative abundance differences in acidic and basic species of Fd+LC fragments indicated potential deamidation or conformational variants.

Benefits and Practical Applications

• High sensitivity and resolution facilitate comprehensive biosimilarity assessment in compliance with regulatory guidelines.
• Workflow flexibility supports both denatured and native analyses at intact and subunit levels.
• Applicable to quality control laboratories for release testing, stability studies, and comparability protocols.

Future Trends and Opportunities

• Integration of automated data analysis pipelines and machine learning for faster interpretation of complex spectra.
• Expansion to top-down proteomics for full sequence coverage and mapping of post-translational modifications.
• Development of high-throughput native MS assays for rapid screening of mAb variants.
• Enhanced mass analyzers and fragmentation methods to further resolve isobaric species and conformers.

Conclusion

The presented intact and subunit RPLC-MS and native IEX-MS workflows on a benchtop QToF platform enable reliable, high-resolution profiling of mAb charge variants and glycoforms. This approach supports thorough comparability assessment of biosimilars, meeting stringent regulatory requirements and facilitating quality control practices.

References

• No explicit literature references were provided in the source document.