Fast profiling of the N-glycan population in biotherapeutic antibodies by UHPLC-FLD with MS confirmation

Importance of N-Glycan Profiling in Antibody Therapeutics

The glycosylation pattern of monoclonal antibodies is a critical quality attribute that influences their efficacy, immunogenicity, and pharmacokinetics. Detailed characterization of N-glycan populations ensures consistent product performance, supports biosimilarity assessments, and aids in meeting regulatory requirements under ICH Q5E/Q6B and USP guidelines.

Objectives and Overview

This study aimed to establish a rapid, robust UHPLC-FLD workflow for comprehensive profiling of 2-AA and 2-AB labeled N-glycans in human serum IgG and a commercial chimeric IgG1 (infliximab). Key goals included:

• Optimizing sample preparation, labeling, and chromatographic separation to increase throughput
• Validating the method on two model antibodies
• Confirming glycan structures via exoglycosidase arrays and high-resolution accurate-mass MS

Methodology and Instrumentation

A streamlined workflow combined denaturation, reduction/alkylation, PNGase F-mediated release, and derivatization with 2-AA or 2-AB. Cleanup was achieved by HILIC SPE on an Accucore 150-Amide column. Separation employed Thermo Scientific Vanquish Horizon UHPLC with fluorescence detection, using three column lengths (50, 150, 250 mm) to balance resolution and speed. Structural confirmation utilized targeted exoglycosidase digests (ABS, BKF, SPG, GUH) followed by offline Q Exactive Plus Hybrid Quadrupole-Orbitrap MS in negative‐ion mode.

Main Results and Discussion

Chromatographic evaluation on human IgG yielded 18 well-resolved peaks on 250 mm and 150 mm columns; the 50 mm column achieved sub-15 min runs with acceptable resolution for simpler profiles. Infliximab analysis produced 24 distinct peaks, resolved effectively on longer columns, and 38 glycan structures were annotated overall, including high-mannose, hybrid, and complex bi-antennary species with varied core fucosylation, galactosylation, bisecting GlcNAc and sialylation. Exoglycosidase digestion arrays enabled unambiguous assignment of linkages and branching patterns.

Benefits and Practical Applications

• High sensitivity FLD detection allows quantitation of minor glycoforms
• Modular column lengths permit flexible throughput vs. resolution trade-offs
• Exoglycosidase arrays combined with HRAM MS deliver rapid structural elucidation
• Compliance with regulatory guidances for biotherapeutic characterization

Future Trends and Opportunities

Continued advances may include integration of on-line MS detection for real-time identification, miniaturized or multiplexed HILIC workflows for ultra-high throughput, and AI-driven data analysis for automated glycan annotation. Emerging labeling chemistries or fluorescent tags could further enhance sensitivity and dynamic range.

Conclusion

The presented UHPLC-FLD method with MS confirmation provides a rapid, versatile, and regulatory-compliant platform for detailed N-glycan profiling of therapeutic antibodies. It supports high-throughput screening, comparability studies, and ensures robust quality control of glycoprotein products.

References

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• ICH Q6B Specifications for Biotechnological Biological Products, 1999