Fast UHPLC Analysis of 2AB Labelled N-Linked Glycans of Human IgG with an Agilent 1290 Infinity LC System

Importance of the Topic

Rapid and precise analysis of N-linked glycans on immunoglobulin G (IgG) is critical in biopharmaceutical development, as glycosylation patterns influence therapeutic efficacy, stability, and immunogenicity. High-throughput glycan profiling supports process optimization, quality control, and regulatory compliance.

Objectives and Study Overview

This study demonstrates a fast UHPLC method using an Agilent 1290 Infinity Quaternary LC System with a sub-2 µm HILIC amide column for the separation of 2-AB labeled human IgG N-linked glycans. The main goals were to reduce analysis time while maintaining high resolution and reproducibility.

Methodology and Instrumentation Used

• Sample: 2-AB labeled human IgG N-glycan library (Prozyme GLYKO GKSB-005).
• System: Agilent 1290 Infinity LC with binary pump (G4220A), high-performance autosampler (G4226A), thermostatted column compartment (G1316C), and fluorescence detector (G1321).
• Column: HILIC Glycan Amide, 2.1 × 150 mm, sub-2 µm particles.
• Mobile phases: 100 mM ammonium formate pH 4.5 (A) and acetonitrile (B).
• Gradient: Start at 75% B, linear to 52.5% B over 18 min, adjust flow between 1.0 and 0.5 mL/min for re-equilibration.
• Temperature: Column held at 60 °C.
• Detection: Fluorescence (Ex 330 nm, Em 420 nm), injection volume 2 µL with needle wash.

Main Results and Discussion

• Eight major human IgG N-glycan peaks and free dye were baseline separated in under 10 minutes.
• Retention time precision and peak area reproducibility were confirmed across six replicates, demonstrating method robustness.
• Elevated column temperature and optimized flow programming enabled the fast cycle without loss of resolution.

Benefits and Practical Applications

• Analysis time reduced by over 65% compared to conventional methods (from ~30 min to <10 min), boosting sample throughput.
• Fluorescence detection ensures high sensitivity for low-abundance glycoforms.
• Suitable for biopharmaceutical QC, comparability studies, and glycoengineering research.

Future Trends and Possibilities

• Coupling with mass spectrometry for detailed structural analysis of glycan isomers.
• Advancements in column technology to further shorten run times.
• Automation of sample preparation and data processing for large-scale glycomics.
• Implementing software tools for automated glycan identification and quantitation.

Conclusion

The described UHPLC-FLD approach on the Agilent 1290 Infinity platform offers a rapid, reproducible, and high-throughput solution for profiling 2-AB labeled N-linked glycans of human IgG, meeting the demands of both research laboratories and industrial quality control.

Reference

Agilent Technologies, Inc. Technical Overview: Fast UHPLC Analysis of 2AB Labelled N-Linked Glycans of Human IgG with an Agilent 1290 Infinity LC System (2014).