HPAE-PAD Analysis of N-linked Oligosaccharides from Glycoproteins Using Dual Eluent Generation Cartridge Mode

Importance of the Topic

High-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) is a critical analytical approach for characterizing N-linked oligosaccharides on glycoprotein therapeutics. Because glycosylation affects drug efficacy, pharmacokinetics, and immunogenicity, reliable mapping of carbohydrate structures ensures product consistency and safety in biopharmaceutical development.

Objectives and Study Overview

The study aimed to evaluate a Dual Eluent Generation Cartridge (Dual EGC) mode on a Thermo Scientific™ Dionex™ ICS-6000 HPIC™ system as an alternative to manual sodium hydroxide/sodium acetate eluent preparation. Using electrolytically generated potassium hydroxide (KOH) and potassium methanesulfonate acid (KMSA), the method was tested on released N-linked oligosaccharides from human IgG and bovine fetuin to assess resolution, precision, and accuracy.

Methodology and Instrumentation

• System configuration: Thermo Scientific Dionex ICS-6000 HPIC with an isocratic pump and dual eluent generator cartridges (EGC 400 MSA and EGC 400 KOH) in series.
• Separation column: Dionex CarboPac PA200 guard (1×50 mm) and analytical (1×250 mm) in 1 mm format at 30 °C.
• Detection: Pulsed amperometric with gold-on-PTFE working electrode, Ag/AgCl reference electrode, following a four-step waveform.
• Sample preparation: PNGase F digestion of human IgG, direct injection of oligosaccharide alditols; fetuin standards purchased from Thermo Fisher.
• Gradient elution: Multi-step KMSA/KOH gradient tailored for IgG and fetuin separations, eliminating manual buffer mixing.
• Data analysis: Thermo Scientific Chromeleon CDS v7.2.8 for retention time and peak area assessments.

Main Findings and Discussion

• Resolution: Comparable separation of neutral and charged IgG oligosaccharides to conventional NaOH/NaOAc methods, with clear peak resolution of G0, G1F(1,6), and Neu5Ac-containing species after neuraminidase treatment.
• Precision and accuracy: Retention time and peak area reproducibility across three consecutive injections showed low variability for both human IgG and bovine fetuin standards.
• Operational benefits: Automated eluent generation reduced manual handling errors, improved reproducibility, and increased instrument uptime by eliminating issues related to sodium acetate purity.

Benefits and Practical Applications

The Dual EGC approach streamlines HPAE-PAD workflows by removing the need for manual eluent preparation and sample derivatization. It delivers robust glycan profiling for biopharmaceutical QC, process monitoring, and comparability studies. Its orthogonality to capillary electrophoresis (CE) and hydrophilic interaction chromatography (HILIC) makes it a complementary tool in comprehensive glycan analysis.

Future Trends and Possibilities

• Integration with automated sample preparation platforms to further reduce hands-on time.
• Extension to other glycoprotein classes and complex glycan mixtures, including O-linked and hybrid structures.
• Advancements in high-throughput and miniaturized column formats to accelerate screening in development pipelines.
• Coupling with mass spectrometry for detailed structural elucidation in hybrid analytical workflows.

Conclusion

The Dual Eluent Generation Cartridge mode on the Dionex ICS-6000 HPIC system provides reliable, high-resolution analysis of N-linked oligosaccharides without manual eluent preparation. It achieves performance equivalent to traditional methods while enhancing reproducibility, accuracy, and operational efficiency, making it well suited for routine glycoprotein analysis in biopharmaceutical environments.

Reference

• Huang, B., Chen, L., Weiss, J., & Rohrer, J. (2018). HPAE-PAD analysis of N-linked oligosaccharides from glycoproteins using dual eluent generation cartridge mode. Thermo Fisher Scientific Application Note PO72773-EN.
• Rohrer, J. S., Basumallick, L., & Hurum, D. C. (2016). Profiling N-linked oligosaccharides from IgG by HPAE-PAD. Glycobiology, 26(6), 582–591.
• Thermo Scientific Technical Note 42 (1997). Glycoprotein Oligosaccharide Analysis Using High-Performance Anion-Exchange Chromatography.