Glycoprotein monosaccharide analysis using HPAE-PAD with manually prepared eluent

Importance of the Topic

Glycosylation critically influences protein structure, function and therapeutic efficacy. Monosaccharide composition analysis is essential for quality control of glycoprotein-based biotherapeutics and biosimilars, ensuring consistency in production and regulatory compliance.

Objectives and Study Overview

This work presents a robust high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) method using manually prepared sodium hydroxide eluent. It aims to accurately quantify six monosaccharides (fucose, galactosamine, glucosamine, galactose, glucose and mannose) in glycoprotein hydrolysates, comparing TFA hydrolysis for neutral sugars and HCl for amino sugars.

Used Methodology

A dual hydrolysis strategy was applied: trifluoroacetic acid (0.3 mg/mL protein) for neutral sugars and 6 M hydrochloric acid for amino sugars, followed by speed-vac drying and reconstitution. Separation employed a CarboPac PA20 analytical column (3×150 mm) with an AminoTrap guard column and inline BorateTrap to remove contaminant borate. Isocratic elution (10 mM NaOH), step gradient (200 mM NaOH) and re-equilibration achieved baseline separation within 13 min. Detection was by pulsed amperometry on a gold working electrode.

Used Instrumentation

• Thermo Scientific Dionex ICS-5000+ RFIC system with DP dual pump and DC detector compartment
• Electrochemical detector and disposable Au working electrode
• CarboPac PA20 analytical column (3×150 mm) with AminoTrap guard (3×30 mm) and BorateTrap inline trap
• AS-AP autosampler with cooling tray
• SpeedVac concentrator with acid trap

Main Results and Discussion

The method achieved baseline resolution of all six monosaccharides in standard and hydrolyzed glycoprotein samples (IgG, fetuin, AGP). Calibration was linear (r2>0.983) over 1.56–300 µM (galactosamine limited to 50 µM). Limits of detection ranged 0.1–0.25 µM. Precision studies showed <2% RSD for retention times and peak areas; intermediate precision <3.4%. Recovery in spiked hydrolysates ranged 80–120%. Robustness tests with ±10% eluent strength, flow rate, temperature yielded minimal impact on resolution and asymmetry.

Benefits and Practical Application of the Method

The assay allows direct carbohydrate detection without derivatization, reducing analysis time, chemical use and analyst exposure. It offers high selectivity, sensitivity and reproducibility, making it suitable for routine QC of biotherapeutics and biosimilar development.

Future Trends and Potential Applications

• Automated eluent generation to streamline workflow
• Hyphenation with mass spectrometry for structure elucidation
• Miniaturized and high-throughput platforms for rapid screening
• Extended application to complex glycans, oligosaccharides and glycopeptides
• Online process monitoring in biomanufacturing

Conclusion

A validated HPAE-PAD method with manually prepared eluent was developed for monosaccharide analysis in glycoproteins. The approach provides rapid, accurate and robust quantification critical for quality control in biopharmaceutical research and production.

Reference

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