An ultrafast, batch-to-batch comparison of monoclonal antibody glycosylation

Significance of the Topic

This application note addresses the critical need for rapid and reliable glycosylation profiling of monoclonal antibodies, a key quality attribute that influences therapeutic efficacy, safety, and stability.

Goals and Study Overview

The study demonstrates a high-throughput HILIC UHPLC method combined with fluorescence detection of 2-aminoanthranilic acid (2-AA) labeled N-glycans and confirmation by accurate-mass mass spectrometry. Rituximab serves as a model monoclonal antibody for proof of concept, focusing on batch-to-batch glycoform comparison with sufficient resolution and greatly reduced analysis time.

Methods and Instrumentation

Sample preparation involves protein denaturation, reduction, alkylation, enzymatic N-glycan release with PNGase F, labeling with 2-AA via reductive amination, and HILIC cleanup.

Instrumentation Used

• Thermo Scientific Vanquish Horizon UHPLC system with Rapid Separation Quaternary Pump, Thermostatted Well-Plate Autosampler, Thermostatted Column Compartment, and Fluorescence Detector
• Accucore 150-Amide-HILIC columns (2.1 × 50 mm and 2.1 × 150 mm, 2.6 µm)
• Thermo Scientific Q Exactive Plus Hybrid Quadrupole-Orbitrap Mass Spectrometer (HESI source)
• Data analysis via Chromeleon 7.2 and Xcalibur 2.2 software

Main Results and Discussion

The optimized UHPLC-FLD method separates the eight major 2-AA-labeled N-glycans of rituximab in 2.5 minutes, representing a 20-fold reduction in run time compared to conventional methods. Retention time precision across multiple injections is excellent, and accurate-mass MS confirms glycan structures. Batch comparison of three rituximab lots reveals consistent overall profiles with minor variations in selected glycoforms.

Benefits and Practical Applications

• High-throughput and reproducible screening of mAb glycosylation
• Significant reduction in analysis time supports faster process development and quality control
• Compatibility with quality-by-design frameworks for biosimilar and biotherapeutic development

Future Trends and Opportunities for Use

Integration of ultrafast glycoprofiling into automated workflows, expansion to other therapeutic glycoproteins, and coupling with advanced MS techniques for deeper structural elucidation can further streamline biopharmaceutical development and in-line process monitoring.

Conclusion

The presented ultrafast HILIC UHPLC-FLD/MS workflow offers rapid, sensitive, and reproducible N-glycan profiling of monoclonal antibodies, making it a powerful tool for bioprocess development, biosimilar comparability, and quality control.

References

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