Fast and Efficient HILIC Methods for Improved Analysis of Complex Glycan Structures

Significance of the Topic

Glycosylation is a critical post-translational modification influencing the efficacy, safety, and half-life of therapeutic proteins, notably monoclonal antibodies. Detailed profiling of glycan structures is essential throughout biotherapeutic development and manufacturing to ensure consistent product quality and performance.

Objectives and Study Overview

This work evaluates a sub-2 µm HILIC amide column for rapid, high-resolution separation of 2-AB labeled N-linked glycans from human IgG1 and bovine fetuin. The goal was to achieve fast analysis times without compromising resolution and to compare performance against a competing sub-2 µm HILIC column.

Methodology and Instrumentation

2-AB labeling followed reductive amination and cleanup steps. Glycan separations used an Agilent AdvanceBio Glycan Mapping HILIC column (2.1 × 150 mm, 1.8 µm) at 55 °C with a gradient of ammonium formate buffer (pH 4.5) and acetonitrile. Fluorescence detection monitored excitation at 260 nm and emission at 430 nm. For mass confirmation, an Agilent 6550 iFunnel Q-TOF LC/MS with Dual JetStream source was employed.

Main Results and Discussion

• A 2-AB dextran ladder was baseline resolved in under 15 minutes.
• Human IgG glycans achieved ultra-high resolution, reducing run time by 40% (to ~9 minutes) compared with a rival column, while improving peak capacity and selectivity.
• Bovine fetuin bi- and triantennary glycans were separated in under 12 minutes, with clear distinction of major structural isomers.
• Q-TOF mass spectra matched theoretical glycan masses within 6 ppm, confirming structural assignments.

Benefits and Practical Applications

These rapid HILIC methods enable high-throughput glycan profiling in biopharmaceutical quality control and research settings. The combination of sub-2 µm packing and optimized gradients delivers enhanced resolution, shorter analysis times, and robust performance for complex glycan mixtures.

Future Trends and Opportunities

• Integration with automated sample preparation to further boost throughput.
• Expansion to other labeling chemistries and glycan types.
• Coupling HILIC separations with high-resolution MS/MS for in-depth structural elucidation.
• Development of dedicated software for automated glycan peak assignment and quantitation.

Conclusion

The Agilent 1.8 µm HILIC amide column demonstrated fast, efficient, and highly selective separations of N-linked glycans from diverse glycoprotein sources. It outperformed a comparable sub-2 µm HILIC column by achieving a 40% faster cycle time while maintaining or improving resolution and peak capacity.

Reference

Martosella J., Potter O., Mancheno D., Liu J.: Fast and Efficient HILIC Methods for Improved Analysis of Complex Glycan Structures, Agilent Technologies Application Note, November 2017.