A Novel HILIC Column for High Speed N-linked Glycan Analysis

Significance of the Topic

N-linked glycosylation of monoclonal antibodies (mAbs) critically influences safety, efficacy, and serum half-life of therapeutic proteins. Rapid, high-resolution glycan profiling is essential for biotherapeutic characterization, QC, and process development.

Objectives and Study Overview

This study evaluates the performance of the Agilent AdvanceBio Glycan Mapping column, featuring novel HILIC amide chemistry on sub-2 µm particles, to achieve faster and more detailed analysis of 2-AB labeled human IgG N-linked glycans. Comparative experiments assess speed, resolution, and stability relative to existing HPLC columns.

Methodology and Instrumentation

The analytical workflow included enzymatic release of N-linked glycans from IgG, fluorescent labeling with 2-aminobenzamide (2-AB), followed by HILIC separation. Two gradient modes were applied:

• Fast mode: sub-10 min isocratic and gradient steps between 75–40% acetonitrile.
• High-resolution mode: extended gradient over 30 min at 55 °C.

Instrumentation details are listed in the Instrumentation section below.

Instrumentation

• Agilent AdvanceBio Glycan Mapping Column, 2.1×150 mm, 1.8 µm.
• Agilent 1290 Infinity LC System.
• Agilent 1260 Infinity Fluorescence Detector (Ex 260 nm/Em 430 nm).
• Mobile phases: 100 mM ammonium formate pH 4.5 and acetonitrile.

Results and Discussion

The new column reduced glycan elution times by ~40%, achieving complete separation in under 10 minutes with baseline resolution (RS > 1.6) and narrow peak widths (~0.06 min). Extended gradients delivered ultra-high resolution (RS ~ 2.6–5.4) and increased peak capacity (~220). Comparisons with a competitor’s column of identical dimensions showed superior speed, resolution, and peak capacity under both fast and ultra-fast conditions.

Longevity testing at 60 °C over 5,000 injections demonstrated stable retention times, consistent peak shape, and maintained sensitivity, confirming excellent column lifetime and chemical robustness.

Benefits and Practical Applications

• High throughput glycan screening for mAb development and QA/QC.
• Enhanced resolution enables detailed profiling of isomeric glycans.
• Reduced analysis time increases laboratory productivity.
• Compatibility with fluorescence and MS detection provides structural insight.

Future Trends and Opportunities

As biotherapeutics diversify, rapid glycan analysis will be vital for novel molecule characterization. Integration with MS, automation of sample prep, and development of tailored HILIC phases may further improve throughput and structural resolution in glycoanalytics.

Conclusion

The Agilent AdvanceBio Glycan Mapping column offers a robust HILIC solution for fast, high-resolution N-linked glycan profiling of mAbs, combining speed, resolution, and stability to support biopharmaceutical research and QC workflows.

References

• R. Jefferis, Biotechnol. Prog. 21, 11 (2005).
• J. N. Arnold et al., Adv. Exp. Med. Biol. 564, 27 (2005).
• R. Abès, J. L. Teillaud, Pharmaceutics 3, 146 (2010).
• L. R. Ruhaak et al., Anal. Bioanal. Chem. 397, 3457 (2009).