HPAE-PAD profiling of N-linked oligosaccharides from glycoproteins using dual eluent generation cartridges

Importance of the Topic

Glycosylation heterogeneity influences therapeutic protein efficacy, stability and immunogenicity. Robust analytical methods to profile N-linked oligosaccharides are essential for biopharmaceutical development, quality assurance and regulatory compliance.

Objectives and Study Overview

This application note evaluates a Dual EGC HPAE-PAD method to streamline eluent preparation and improve reproducibility in N-linked glycan profiling. Model glycoproteins—human serum IgG and α1-acid glycoprotein (AGP)—were released, purified and analyzed to compare performance against traditional sodium hydroxide/acetate gradients.

Methodology and Instrumentation

The method employs a Thermo Scientific Dionex ICS-6000 HPIC system configured in Dual EGC mode, using RFIC cartridges for methanesulfonic acid (MSA) and potassium hydroxide (KOH) in series. Potassium methanesulfonate/potassium hydroxide (KMSA/KOH) gradients are generated electrolytically and delivered through a CarboPac PA200 1 mm column at 0.063 mL/min. Glycan release was achieved by Rapid PNGase F digestion with and without denaturation, followed by HyperSep Hypercarb SPE cleanup. Exoglycosidase digestions (neuraminidase, fucosidase) and standard glycans supported peak identification. Chromeleon CDS software managed gradient control and cartridge lifetime.

Key Results and Discussion

• High-resolution separation of neutral and sialylated N-linked glycans from IgG and AGP matched or exceeded traditional methods.
• Neuraminidase and fucosidase treatments confirmed glycan assignments and demonstrated selective linkage resolution.
• Vendor-dependent AGP profiles revealed glycoform heterogeneity in complex tetra-antennary structures.
• Precision overlays of repeated injections showed excellent retention time and peak area reproducibility.

Benefits and Practical Applications

Automated eluent generation eliminates manual preparation errors, reduces contamination risk, and accelerates method setup. The dual EGC approach enhances day-to-day consistency and operational efficiency, making it well suited for routine glycan monitoring in QA/QC and process development laboratories.

Future Trends and Potential Applications

Integration of Dual EGC HPAE-PAD with mass spectrometry could enable detailed structural and quantitative glycomics. Further miniaturization, high-throughput formats and advanced software-based eluent optimization will expand applications to novel glycoprotein therapeutics and biomarker discovery.

Conclusion

The dual electrolytic eluent generation system on the Dionex ICS-6000 provides reliable, high-purity KMSA/KOH gradients for efficient N-linked glycan profiling. Comparable resolution to manual eluents, along with improved reproducibility and reduced hands-on time, underscores its value for biopharmaceutical glycan analysis.

Reference

1. Rohrer JS et al. Glycobiology 2016, 26, 585–591.
2. Thermo Fisher Scientific. Dionex ICS-6000 Dual EGC Installation Guide, Doc No 065760-01, 2018.
3. Thermo Fisher Scientific. Dionex ICS-6000 Operation Manual, Doc No 22181-97002, 2018.
4. Thermo Fisher Scientific Technical Note 110: Carbohydrate Determination by HPAE-PAD, 2011.
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6. Rohrer JS. Glycobiology 1995, 5, 359–360.
7. Thermo Scientific Application Update 72829, 2018.