Charge Profiling of 2AB-labelled N-linked Glycans

Importance of the Topic

The glycosylation charge profile of therapeutic glycoproteins is critical for ensuring product safety, efficacy, and batch consistency. Charged N-linked glycans, especially those containing sialic acids, influence pharmacokinetics and immunogenicity. Accurate profiling of glycan charge variants supports quality control in biopharmaceutical production.

Objectives and Overview

This application study aimed to separate and quantify 2-AB labeled N-glycans released from bovine fetuin, resolving species with zero to five sialic acids. The analysis employed strong anion-exchange chromatography coupled with fluorescence detection on an Agilent 1260 Infinity Bio-inert Quaternary LC System.

Methodology and Instrumentation

The workflow involved enzymatic release of N-glycans using PNGase F, 2-AB labeling, and cleanup via HILIC cartridges. Separation was performed on an Agilent Bio SAX column (2.1 × 250 mm, 5 µm) using a gradient from aqueous buffer to ammonium formate (pH 4.5) in 20% acetonitrile. Key parameters:

• Mobile phases: A) 20% acetonitrile/water; B) 20% acetonitrile/250 mM ammonium formate, pH 4.5
• Flow rate: 0.2 mL/min; injection volume: 0.5 µL
• Fluorescence detection: excitation 260 nm, emission 430 nm
• Autosampler at 5 °C; column at ambient temperature

Used Instrumentation

• Agilent 1260 Infinity Bio-inert Quaternary Pump (G5611A)
• Agilent 1260 Infinity High-performance Bio-inert Autosampler (G5667A)
• Agilent 1290 Infinity Thermostat (G1330B)
• Agilent 1290 Infinity Thermostatted Column Compartment with bio-inert heat exchangers (G1316C; G5616-81000)
• Agilent 1260 Infinity Fluorescence Detector (G1321B)
• Software: Agilent OpenLAB CDS ChemStation Edition C.01.06

Main Results and Discussion

Six distinct peaks corresponding to neutral, mono-, di-, tri-, tetra-, and penta-sialylated glycans were baseline separated. Retention time reproducibility showed RSD < 0.002%, and peak area precision was better than 10% RSD. The charge variants matched expected glycan compositions based on known fetuin structures.

Benefits and Practical Applications

• High resolution separation of charged glycan variants enables detailed profiling required in biopharmaceutical quality control.
• Fluorescence detection of 2-AB labels provides high sensitivity and specificity.
• Bio-inert LC system minimizes sample interaction, improving accuracy for labile sialylated species.

Future Trends and Applications

Emerging glycan analysis techniques may integrate mass spectrometry for structural elucidation of charge variants. Advances in automation and microfluidic platforms could increase throughput. Enzymatic or chemical derivatization methods may expand detection of other anionic modifications such as phosphorylation.

Conclusion

This study demonstrates robust charge profiling of 2-AB labeled N-glycans using anion-exchange LC with fluorescence detection, achieving excellent resolution and precision for up to five sialic acids. The approach is well suited for routine monitoring of glycosylation charge variants in biotherapeutic development.

References

1. Gil GC et al. High Throughput Quantification of N-Glycans Using One-Pot Sialic Acid Modification and MALDI-TOF MS. Anal Chem 2010, 82:6613–6620.
2. Schneider S. Sensitive and reproducible glycan analysis of human Immunoglobulin G. Agilent Technologies Application Note, 5991-4801EN, 2014.
3. Fernandes D. Biopharmaceutical Sialylation. Eur Biopharm Rev 2006, Spring:100–104.