Characterization of Released N-linked Glycans in Biosimilar mAb Drug Products Using the Xevo™ G3 QTof

Significance of the Topic

Characterization of N-linked glycans on monoclonal antibodies (mAbs) is critical for ensuring therapeutic safety and efficacy, especially as biosimilar products enter the market. Glycan structures influence immunogenicity, pharmacokinetics, and receptor interactions. A robust analytic workflow capable of resolving low-abundance, structurally complex glycans is essential for demonstrating comparability between an innovator mAb and its biosimilar.

Objectives and Overview of the Study

This study aimed to establish a streamlined, high-sensitivity workflow to profile released N-linked glycans from the innovator infliximab (Remicade™) and its biosimilar (Renflexis™). The objectives were:

• To develop a reproducible sample preparation and labeling procedure for N-glycans.
• To achieve chromatographic separation and sensitive detection of glycan variants.
• To leverage mass spectrometry (MS) for structural confirmation and relative quantification.
• To compare glycan profiles between the originator and biosimilar batches for biosimilarity assessment.

Methodology and Used Instrumentation

Sample preparation involved enzymatic release of N-glycans followed by labeling with GlycoWorks RapiFluor-MS on an automated pipetting robot. Separation was conducted by HILIC UPLC on an ACQUITY Premier Glycan BEH Amide column at 60 °C, with inline fluorescence detection (λexc=265 nm, λem=425 nm). MS analysis employed a Xevo™ G3 QTof with ESI positive mode, low/high collisional energy acquisition (MSE) or data-dependent MS/MS to generate precursor and diagnostic fragment ions (100–2000 m/z). Data were processed in the waters_connect informatics platform using the UNIFI App and an RFMS-glycan library for automated glycan identification and quantitation.

Main Results and Discussion

Comparative profiles revealed distinct glycovariant patterns between the originator and biosimilar:

• Sialylated glycans exhibited differing NeuAc/NeuGc ratios, reflecting cell-line origin (CHO vs murine).
• High-mannose species (M5, M6) and α-galactosylated forms showed variable abundances.
• Elevated-energy fragment data provided structural validation, even for low-abundance species (~1 % relative level).
• MSE workflows enabled simultaneous capture of precursor and fragment spectra; targeted DDA improved clarity for coeluting or isomeric glycans.

These findings underscore the workflow’s capacity to distinguish compositional and linkage differences that impact biosimilar evaluation.

Benefits and Practical Applications of the Method

• High sensitivity fluorescence detection coupled with RFMS labeling ensures detection of trace glycoforms.
• Accurate mass measurement and collision-induced fragmentation deliver confident structural assignments.
• Automated data processing accelerates biosimilarity assessments.
• Inline UPLC-MS analysis supports routine QC and comparability studies for therapeutic mAbs.

Future Trends and Potential Applications

Integration of ion mobility separation and advanced informatics will further refine isomer resolution and stereochemical assignments. Machine-learning-driven data processing may enable predictive modeling of glycosylation profiles, supporting process optimization in biologics manufacturing. Expanded glycan libraries and standardized retention indices will enhance cross-laboratory comparability.

Conclusion

This workflow—combining GlycoWorks RapiFluor-MS labeling, HILIC UPLC, Xevo G3 QTof MS, and waters_connect informatics—provides a sensitive, high-throughput approach to characterize released N-linked glycans in mAb products. It effectively differentiates glycan profiles between innovator and biosimilar infliximab, supporting rigorous biosimilarity evaluations and ensuring product quality.

References

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