New AdvanceBio Workflow: Glycan Mapping Columns, Sample Preparation, and Standards

Importance of the Topic

Glycan mapping plays a critical role in the characterization and quality control of biotherapeutics. Detailed profiling of N-linked glycans influences drug efficacy, immunogenicity and regulatory compliance. An end-to-end workflow that ensures accuracy and reproducibility is essential for rapid biopharmaceutical development.

Objectives and Study Overview

This document presents the Agilent AdvanceBio glycan mapping solution, comprising optimized columns, sample preparation kits and standards. It aims to demonstrate enhanced resolution, speed and consistency for 2-AB labeled N-glycan analysis from glycoprotein samples.

Methodology

• Deglycosylation: Enzymatic release of N-linked glycans using PNGase F under denaturing conditions.
• Derivatization: Labeling of released glycans with 2-aminobenzamide to enable fluorescence detection.
• Cleanup: Solid-phase extraction cartridges for removal of excess reagents and salts.
• Chromatography: HILIC separation of labeled glycans under gradient elution with ammonium formate buffer and acetonitrile.

Instrumentation

• Columns: AdvanceBio glycan mapping columns available in 1.8 μm UHPLC formats (up to 1200 bar) and 2.7 μm superficially porous particles (up to 600 bar).
• LC System: Agilent 1290 Infinity series coupled to a fluorescence detector with excitation at 260 nm and emission at 430 nm.
• Conditions: Flow rates from 0.25 to 0.5 mL/min, column temperatures of 55–60 °C, sample thermostat at 6–10 °C, injection volumes around 2 μL.

Results and Discussion

The AdvanceBio solution achieved complete separation of up to 18 distinct glycan peaks in under ten minutes, representing a 40 percent time reduction versus competitive media. The 1.8 μm columns delivered narrower, symmetric peaks at high pressures, while the 2.7 μm superficially porous format offered high resolution at pressures below 400 bar, enabling compatibility with standard HPLC systems. Use of dextran ladder and IgG glycan standards ensured retention mapping and verified method reproducibility.

Benefits and Practical Applications

• Streamlined workflow with single-source kits reduces vendor complexity and preparation time.
• High-throughput analysis supports accelerated process development and scale-up.
• Robust reproducibility facilitates biosimilar comparability studies and routine QA/QC.
• Compatibility with fluorescence and mass spectrometric detection enables structural confirmation.

Future Trends and Applications

Integration of automation and robotics for sample preparation will further increase throughput. Emerging glycoengineering in new biological modalities will drive demand for deeper glycan structural analysis. Advances in high-resolution MS coupling and computational glycomics are expected to expand glycan mapping capabilities.

Conclusion

The Agilent AdvanceBio glycan mapping solution provides a comprehensive, high-performance workflow for rapid, reproducible N-glycan profiling. Its combination of optimized sample preparation, high-resolution columns and reliable standards enhances laboratory efficiency and data consistency.

References

• Agilent Technologies. AdvanceBio glycan mapping solution whitepaper. May 2015.