Comprehensive Biosimilar Comparability Assessment via Intact and Subunit RP-MS and IEX-UV-MS Using the Xevo™ G3 QTof System

Importance of the Topic

The rapid growth of biosimilar monoclonal antibody (mAb) development demands reliable analytical workflows for detailed comparability assessment. Small variations in post-translational modifications and charge variants can impact drug safety and efficacy. Combining orthogonal liquid chromatography-mass spectrometry (LC-MS) techniques at both intact and subunit levels enhances confidence in biosimilar characterization and supports regulatory submissions.

Objectives and Study Overview

This study evaluates the performance of the Xevo G3 QTof system coupled with an ACQUITY Premier UPLC and waters_connect informatics for comprehensive comparability testing of infliximab innovator (Remicade) and three biosimilars (Inflectra, Avsola, Renflexis). Key goals:

• Compare intact mass profiles via reversed-phase (RP) LC-MS.
• Localize variants through subunit analysis after IdeS and carboxypeptidase B digestion.
• Profile charge variants using native ion-exchange (IEX) UV-MS.
• Demonstrate streamlined untargeted and targeted data processing with UNIFI and INTACT Mass applications.

Methodology and Instrumentation

Samples of intact mAbs, IdeS-digested subunits, and carboxypeptidase B-treated products were analyzed under denaturing RP and native IEX conditions. Key parameters included:

• ACQUITY Premier Protein BEH C4 column at 80 °C for intact/subunit RP separations.
• BioResolve SCX mAb column at 30 °C for native IEX separations.
• Xevo G3 QTof operated in positive mode, m/z ranges 50–5000 (RP) and 400–8000 (IEX).
• Intelligent Data Capture for noise reduction and reduced file size.
• Data processed via waters_connect using UNIFI App and INTACT Mass workflow.

Used Instrumentation

• Xevo G3 QTof mass spectrometer
• ACQUITY Premier UPLC system
• ACQUITY UPLC Tunable UV Detector (280 nm)
• ACQUITY Premier Protein BEH C4 column (2.1×50 mm, 1.7 µm)
• BioResolve SCX mAb column (2.1×100 mm, 3 µm)
• waters_connect informatics with UNIFI and INTACT Mass apps

Main Results and Discussion

Reversed-phase LC-MS of intact mAbs revealed differences in C-terminal lysine variants and relative N-glycoform distributions. After removal of lysine via carboxypeptidase B, minor variations in major N-glycoforms were observed. Subunit analysis localized immunogenic N-glycolylneuraminic acid–containing glycans in Remicade and Inflectra, correlating to their Sp2/0 cell-line origin, while CHO-expressed Avsola and Renflexis lacked these forms. Light chain and Fd glycation levels were below 0.5% for Remicade but rose to 2–3% for biosimilars.

Native IEX-UV-MS charge profiles showed consistent quantitation of main, basic (C-terminal lysine) and acidic variants. MS detection under each peak enabled confident assignment of deamidation, glycoforms, and lysine processing. Subunit IEX-MS distinguished Fd+LC variants and Fc species, offering a rapid screening alternative to peptide mapping for CDR modifications.

INTACT Mass untargeted workflows provided an automated dashboard for rapid comparison of deconvoluted mass lists, highlighting lower variant complexity in Renflexis versus Remicade.

Benefits and Practical Application

• Orthogonal RP-LC-MS and IEX-UV-MS deliver complementary data on mass, glycosylation, charge variants, and CDR integrity.
• Streamlined sample preparation and intelligent data capture shorten analysis time and reduce data volume.
• waters_connect platform supports GxP compliance and high-throughput screening with both discovery and targeted workflows.
• Subunit IEX-UV-MS offers a rapid first-pass for CDR deamidation assessment ahead of in-depth peptide mapping.

Future Trends and Opportunities

Advances in high-resolution MS and AI-driven data processing are poised to further automate biosimilar comparability workflows. Integration of native MS techniques with multi-attribute monitoring will enhance real-time release testing. Expanded use of cloud-based informatics may enable global data sharing and collaborative quality assessments across manufacturing sites.

Conclusion

The Xevo G3 QTof system combined with ACQUITY Premier UPLC and waters_connect informatics provides a robust, compliant, and efficient platform for intact and subunit RP-MS and IEX-UV-MS analyses. Orthogonal data confirm key differences in C-terminal lysine variants, glycoform profiles, and charge variants among infliximab biosimilars, supporting detailed comparability that meets regulatory expectations.

References

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