Monosaccharide and Sialic Acid Determinations in Biosimilars Using HPAE-PAD

Importance of the Topic

Therapeutic glycoproteins often feature complex N-glycosylation that modulates their biological activity, serum half-life and immunogenic potential.
Accurate characterization of monosaccharide and sialic acid composition is essential for biosimilar development and quality control.

Objectives and Study Overview

This summary reviews HPAE-PAD methodologies for direct analysis of glycoprotein monosaccharides and sialic acids.
Key applications include screening of sialylation, profiling glycosylation in limited-quantity samples, and ensuring batch-to-batch consistency in biosimilar manufacturing.

Methodology and Instrumentation

HPAE-PAD separates underivatized carbohydrates on high-pH anion-exchange columns.
Analytes are detected via pulsed amperometric detection using disposable Au/PTFE electrodes.
Sample preparation typically involves acid hydrolysis (TFA or HCl) or enzymatic release of N-linked glycans.
Eluents are generated electrolytically (EGC III KOH cartridge) with CR-ATC anion trap columns to produce reagent-free gradients.
Gradient methods employ KOH and sodium acetate in NaOH for sialic acid separations.

Used Instrumentation

Thermo Scientific Dionex ICS-5000+ HPIC and ICS-4000 Capillary HPIC systems
Dionex CarboPac PA20 Analytical & Fast Sialic Acid columns with AminoTrap guard columns
Thermo Scientific Dionex EGC III KOH Eluent Generation Cartridge and CR-ATC Anion Trap Column
Carbohydrate PTFE disposable Au working electrodes with Ag/AgCl reference cell
Flow rates: 0.5 mL/min on analytical columns; 0.4 µL injections on capillary systems.

Main Results and Discussion

Monosaccharide profiling of IgG hydrolysates achieved baseline separation of fucose, glucosamine, galactose, glucose and mannose with high reproducibility over hundreds of runs.
Sialic acid screening distinguished Neu5Ac and Neu5Gc in fetuin and AGP, demonstrating sensitivity to pmol levels and eliminating derivatization artifacts.
Analysis of PSA and transferrin glycosylation used <10 µg sample, revealing consistent monosaccharide ratios and enabling detection of N- and potential O-linked glycans.
A 16.5 min gradient method separated sialic acids in glycoprotein hydrolysates with increased throughput compared to traditional HPLC assays.

Benefits and Practical Applications

Direct detection without derivatization reduces sample preparation time, chemical waste and analyst exposure.
High selectivity and sensitivity support low-level analyte quantification and minor glycosylation variant detection.
Reagent-free eluent generation ensures consistent, automatable workflows.
Capillary HPAE-PAD minimizes sample and eluent consumption for biomarker discovery in limited clinical specimens.

Future Trends and Opportunities

Integration of HPAE-PAD with mass spectrometry for simultaneous glycan structure and composition analysis.
Development of faster column chemistries and miniaturized systems to further increase throughput.
Automated sample preparation and on-line hydrolysis/digestion modules for end-to-end glycoprotein characterization.
Expansion of capillary and microfluidic HPAE-PAD platforms in clinical and bioprocess monitoring applications.

Conclusion

HPAE-PAD provides a robust, direct and highly sensitive platform for carbohydrate profiling in glycoprotein therapeutics.
Its proven precision, throughput and reagent-free operation make it well suited for regulatory compliance and biosimilar quality control workflows.

Reference

Thermo Fisher Scientific. Glycoprotein Monosaccharide Analysis Using HPAE-PAD with Eluent Generation. Technical Note 40. 2014.
Thermo Fisher Scientific. Rapid Screening of Sialic Acids in Glycoproteins by HPAE-PAD. Application Update 181. 2014.
Thermo Fisher Scientific. Evaluating Protein Glycosylation in Limited-Quantity Samples by HPAE-PAD. Application Note 1050. 2014.
Thermo Fisher Scientific. Direct Determination of Sialic Acids in Glycoprotein Hydrolyzates by HPAE-PAD. Application Update 180. 2014.