Analysis of glycan Shim-pack GIST-HP Amide (Metal free)

Importance of the Topic

Glycan profiling of monoclonal antibodies plays a critical role in ensuring product quality safety and efficacy in biopharmaceutical development and manufacturing. Detailed information on glycan composition supports consistency in therapeutic performance and regulatory compliance.

Objectives and Study Overview

This application note demonstrates the separation and detection of eight fluorescently labeled N-glycan structures derived from an antibody digest. The primary aim is to showcase the performance of a metal-free amide HILIC column for high-resolution glycan analysis under fluorescence detection.

Methodology

The method employs hydrophilic interaction liquid chromatography under a gradient of ammonium formate buffer and acetonitrile. Glycan species are separated over 62 minutes with a gradual decrease in organic content, achieving baseline resolution of critical isomeric structures. Key parameters include a column temperature of 40 °C a flow rate of 0.5 mL per minute and a 1 µL injection volume.

Instrumentation Used

• Liquid chromatograph: Nexera XR system
• Column: Shim-pack GIST-HP Amide metal-free 100 mm × 2.1 mm I.D. 1.9 μm
• Detector: RF-20Axs fluorescence detector with semi-micro cell (Ex 330 nm Em 420 nm)

Key Results and Discussion

The optimized HILIC method resolved eight common antibody glycan species including G0 GN G0F Man5 G1 G1F Man6 and G2Ft. Sharp peak shapes and consistent retention times were observed reflecting minimal metal-ion interactions in the stationary phase. The fluorescence detection provided high sensitivity for low-level glycan monitoring.

Benefits and Practical Applications

• Enhanced resolution of isomeric glycans improves structural characterization
• Metal-free column chemistry reduces peak tailing and analyte adsorption
• Fluorescence detection delivers sensitive monitoring of derivatized glycans
• Method supports QC laboratories in biopharmaceutical manufacturing

Future Trends and Potential Applications

Continued advancements in HILIC stationary phases may further improve throughput and robustness for glycan mapping. Integration with mass spectrometry detection can expand structural elucidation capabilities. Automated sample preparation workflows and multi-attribute methods will streamline glycoform profiling in process development.

Conclusion

The Shim-pack GIST-HP Amide column demonstrated reliable and high-resolution separation of fluorescently labeled antibody glycans under HILIC conditions. Combined with fluorescence detection, this method offers a robust solution for routine glycan analysis in biopharmaceutical quality control.

References

• Shimadzu Application News 01-00728 JP ENG First Edition Sep 2024