Combining RapiFluor-MS and UNIFI Scientific Information System for a Total N-linked Glycan Solution for Innovator vs. Biosimilar Infliximab Comparisons

Significance of the topic

Glycosylation of monoclonal antibodies plays a critical role in therapeutic function, including antibody-dependent cellular cytotoxicity and pharmacokinetics. As patents expire on innovator mAbs, biosimilar development demands rigorous glycan profiling to ensure structural and functional equivalence.

Objectives and Study Overview

This study presents a streamlined, end-to-end N-linked glycan analysis workflow combining fast sample preparation, high-resolution LC-FLR-MS analysis, and automated data processing. Innovator infliximab (Remicade®) and a biosimilar (Inflectra®) were compared to evaluate the method’s capability for biosimilarity assessment.

Methodology

Sample preparation was performed in under one hour using the GlycoWorks RapiFluor-MS N-Glycan Kit: mAb denaturation, rapid PNGase F release, RFMS labeling in glycosylaminyl form, followed by HILIC μ-elution cleanup. Chromatography used an ACQUITY UPLC Glycan BEH Amide column (2.1×150 mm, 1.7 μm) at 60 °C with a gradient of ammonium formate (pH 4.4) and acetonitrile. Fluorescence detection was set at Ex 265 nm/Em 425 nm. Mass spectrometry employed a Xevo G2-XS QTof in positive mode with lockmass calibration (Glu-Fibrinopeptide B). Data acquisition and processing utilized UNIFI Scientific Information System with GU calibration via a dextran ladder, automated library searches (ΔGU 0.2), comparative analysis tools, and customizable reporting.

Used Instrumentation

• GlycoWorks RapiFluor-MS N-Glycan Kit
• ACQUITY UPLC H-Class Bio System with Glycan BEH Amide column
• Xevo G2-XS QTof Mass Spectrometer
• UNIFI Scientific Information System with Glycan Application Solution and GU library

Main Results and Discussion

Retention times were normalized to glucose units, minimizing inter-run variability. Library searching identified 23 mass-confirmed glycans in Remicade® and 22 in Inflectra®. Stacked FLR chromatograms showed high qualitative similarity, while difference plots revealed slight increases in NeuGc-terminated species and specific antennary galactosylated structures in the biosimilar. Quantitative summary plots (% amount) confirmed these trends with RSDs < 1%, demonstrating robust reproducibility.

Benefits and Practical Applications

This total workflow accelerates glycan profiling with less than one-hour sample prep and enhanced detection of low-abundance and sialylated species. Automated GU calibration and library searches enable non-specialists to perform detailed glycan analysis, supporting QA/QC in biosimilar development, bioprocess monitoring, and regulatory comparability studies.

Future Trends and Potential Applications

Future developments include expansion of comprehensive RFMS-labeled glycan GU libraries, integration of deeper structural elucidation tools, automated high-throughput workflows, and application to a wider range of biotherapeutics, including fusion proteins and complex glycoproteins. Machine-learning based spectral interpretation may further enhance glycan identification and quantitation.

Conclusion

The combined RapiFluor-MS, UPLC H-Class Bio, Xevo G2-XS QTof, and UNIFI platform provides a robust, rapid, and user-friendly solution for N-linked glycan analysis. It streamlines sample prep, improves sensitivity, and offers automated data processing and reporting, facilitating accurate biosimilar comparability and accelerating biopharmaceutical development.

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