N-Glycan analysis of monoclonal antibodies and other glycoproteins using UHPLC with fluorescence detection

Significance of the Topic

Protein glycosylation is one of the most common post-translational modifications, critically influencing stability, activity and immunogenicity of biopharmaceuticals. In particular, N-linked glycans on immunoglobulin G (IgG) antibodies are key determinants of effector function, serum half-life and safety profile. Detailed characterization of glycan heterogeneity is therefore essential for quality control and optimization of therapeutic glycoproteins.

Objectives and Study Overview

This application note demonstrates a robust workflow for analysis of N-linked glycans released from monoclonal antibodies (mAbs) and two avian egg white glycoproteins (ovalbumin and conalbumin). The goals are to achieve high-resolution separation, sensitive fluorescence detection and confident identification of major glycan species using hydrophilic interaction chromatography (HILIC) coupled with UHPLC and fluorescence detection, complemented by accurate-mass ESI-QTOF MS.

Methodology and Instrumentation

Enzymatic release and labeling

• PNGase F digestion of glycoproteins to release N-glycans at 37 °C for 3 h.
• Reductive amination with 2-aminobenzamide (2-AB) at 65 °C for 3 h, introducing a +119 Da tag for fluorescence and MS.
• HILIC-SPE cleanup to remove excess label and salts.

Chromatography and detection

• Agilent 1260 Infinity Bio-inert Quaternary LC System with HILIC Glycan Amide column (2.1×150 mm, 2 µm).
• Fluorescence detection at Ex 260 nm / Em 430 nm for enhanced sensitivity.
• Gradient elution using 100 mM ammonium formate pH 4.5 (A) and acetonitrile (B), optimized separately for mAbs and egg glycoproteins.

Mass spectrometry confirmation

• Agilent 6530 Accurate-Mass Q-TOF LC/MS for compositional assignment.
• Data analysis with Agilent OpenLAB CDS and MassHunter Workstation.

Main Results and Discussion

Monoclonal antibody glycan profiling resolved all major mAb N-glycans, including G0, G0F, Man5, two G1F isomers, G2F and sialylated forms (G2FS1, G2FS2), with excellent peak shape and signal-to-noise. Over 30 distinct peaks were detected in ovalbumin and conalbumin, reflecting complex high-mannose and hybrid structures at single glycosylation sites. MS confirmation aligned observed masses with known glycan compositions, validating the workflow.

Benefits and Practical Applications

• High resolution and sensitivity enable detailed glycoform profiling for biopharmaceutical QC and comparability studies.
• Fluorescence labeling with 2-AB offers robust quantitation and low detection limits.
• Bio-inert UHPLC hardware minimizes sample adsorption and carryover.

Future Trends and Opportunities

Advances expected include integration with glycan retention libraries and automated annotation tools, extension to O-glycan and glycosphingolipid analysis, and higher-throughput platforms. Coupling HILIC separation to tandem MS/MS will further elucidate structural isomers and linkage information, supporting deeper glycomics studies and precision biotherapeutic design.

Conclusion

The described HILIC-UHPLC-FLD workflow using the Agilent 1260 Infinity Bio-inert Quaternary LC System combined with sensitive fluorescence detection and accurate-mass MS provides an effective solution for comprehensive N-glycan analysis of mAbs and other glycoproteins.

Reference

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