Direct Determination of Sialic Acids in Glycoprotein Hydrolyzates by HPAE-PAD

Significance of the Topic

Sialic acids, particularly N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc), are critical terminal residues on glycoproteins that influence their biological activity, stability, pharmacokinetics, and immunogenicity. Accurate quantification of these residues is essential in biopharmaceutical development and quality control, since incorporation of non-human sialic acids such as Neu5Gc can trigger adverse immune responses.

Objectives and Overview

This study aims to develop and validate a direct, sensitive, and rapid HPAE-PAD method for quantifying Neu5Ac and Neu5Gc released from glycoproteins by acid hydrolysis or enzymatic digestion. Five representative glycoproteins—calf fetuin, bovine apo-transferrin, human transferrin, human α1-acid glycoprotein, and sheep α1-acid glycoprotein—were analyzed to demonstrate method applicability.

Methodology and Instrumentation

Sample Preparation:

• Acid hydrolysis: Glycoproteins were treated with 2 M acetic acid at elevated temperature, followed by lyophilization and reconstitution.
• Neuraminidase digestion: Enzymatic release of sialic acids using α-neuraminidase in acetate buffer at 37 °C.

Chromatographic Conditions:

• Instrument: Dionex ICS-3000/5000 system with pulsed amperometric detection and disposable gold on PTFE working electrode.
• Column: CarboPac PA20 (3 × 150 mm) with PA20 guard (3 × 30 mm).
• Eluent: Gradient of sodium acetate (70–300 mM) in 100 mM NaOH over 9.5 min, followed by equilibration.
• Flow rate: 0.5 mL/min; temperature: 30 °C; injection volume: 10 µL.

Main Results and Discussion

The method achieved baseline separation of Neu5Ac and Neu5Gc in under 10 min, with a total cycle time of 16.5 min. Calibration curves were linear over 1–100 pmol Neu5Ac and 0.39–7.8 pmol Neu5Gc (r2 > 0.999). Limits of detection were 0.17 pmol (Neu5Ac) and 0.08 pmol (Neu5Gc), with limits of quantification of 0.50 pmol and 0.30 pmol, respectively. Peak area precision (RSD) was ≤2.8%, and retention time RSD was <0.2%. Recovery studies in spiked hydrolyzates showed 76–102% recovery by dilution and 75–86% after lyophilization. Sample stability at –40 °C for two weeks produced <15% variation. Comparison of acid hydrolysis versus enzymatic digestion highlighted potential under-release or degradation by acid, indicating the need for optimized release conditions.

Benefits and Practical Applications

• Direct analysis without fluorescent derivatization reduces sample handling and preparation time.
• Disposable Au/PTFE electrodes ensure consistent response and simplified maintenance.
• Rapid analysis and reduced eluent consumption support high sample throughput.
• High sensitivity enables trace-level detection in quality control of therapeutic glycoproteins.

Future Trends and Potential Applications

• Further optimization of hydrolysis protocols to maximize sialic acid recovery while minimizing degradation.
• Extension of the method to other sialic acid variants and complex glycan profiling.
• Integration with mass spectrometric detection for enhanced structural characterization.
• Automation and high-throughput sampling for biopharmaceutical process monitoring.

Conclusion

The presented HPAE-PAD method provides a rapid, sensitive, and direct approach for quantifying Neu5Ac and Neu5Gc in glycoprotein samples, facilitating robust monitoring of sialylation in biotherapeutic development and quality control.

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