Determination of glycoprotein sialic acid composition using HPAE-PAD in dual eluent generation cartridge mode

Significance of the topic

Glycoprotein sialic acids, including N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc), influence therapeutic protein stability, activity, and immunogenicity. Accurate quantification of these residues is critical for ensuring drug safety and efficacy, particularly because humans may mount immune responses to Neu5Gc.

Study goals and overview

This work adapted the established Thermo Scientific AU 180 HPAE-PAD method for sialic acid analysis to a miniaturized 1 mm CarboPac PA20 column configuration. The objective was to implement automated dual eluent generation cartridge (Dual EGC) mode for KOH/KMSA gradient delivery, reducing manual eluent preparation and solvent use while maintaining analytical performance.

Methodology and instrumentation

Sample preparation involved acid hydrolysis of glycoproteins (bovine apo-transferrin, calf fetuin, human α1-acid glycoprotein) using 4 M acetic acid at 80 °C for 3 h, followed by drying and resuspension. Calibration standards of Neu5Ac and Neu5Gc (eight and seven levels, respectively) were prepared by serial dilution. Quantification employed high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD).

• Chromatographic system: Thermo Scientific Dionex ICS-6000 HPIC with DP or SP pump, EG eluent generator, DC detector, and AS-AP autosampler.
• Eluent generation: Dual EGC 400 cartridges for KOH and methanesulfonic acid (MSA) to produce a reproducible KOH/KMSA gradient.
• Column: Dionex CarboPac PA20 Guard (1×30 mm) and Separation (1×150 mm) with 0.056 mL/min flow at 30 °C.
• Detection: Thermo Scientific Dionex HPIC electrochemical detector with gold disposable electrode and carbohydrate waveform.

Main results and discussion

The Dual EGC configuration yielded complete separation of Neu5Ac (t_R ≈4 min) and Neu5Gc (t_R ≈14 min) with clear baseline resolution from early eluting matrix components. Calibration curves exhibited excellent linearity (r² >0.9996). Limits of detection were 20.9 nM for Neu5Ac (8.36 fmol) and 10.3 nM for Neu5Gc (4.13 fmol). Method precision (inter- and intra-day) showed RSD <1.1% for peak areas and <0.2% for retention times. Spike recovery of sialic acids ranged from 95.1% to 105%, demonstrating accuracy.

Benefits and practical applications

The Dual EGC approach eliminates manual eluent preparation, reduces solvent waste by ninefold relative to 3 mm columns, and ensures high reproducibility and ease of operation. This streamlined workflow is well suited for biopharmaceutical QA/QC laboratories monitoring glycoprotein sialylation.

Future trends and opportunities

Integration of miniaturized HPAE-PAD methods with automated sample handling and online mass spectrometric detection could further enhance throughput and structural characterization of sialylated glycans. Advances in cartridge lifetime, gradient flexibility, and coupling to multidimensional chromatography will expand applications in glycomics and biotherapeutic development.

Conclusion

The study successfully demonstrated that the AU 180 HPAE-PAD method for glycoprotein sialic acid analysis can be implemented on a 1 mm CarboPac PA20 column using Dual EGC mode. The new configuration provides equivalent separation, sensitivity, and accuracy while minimizing eluent consumption and manual steps.

References

1. Varki A. Sialic acids in human health and disease. Trends Mol Med. 2008;14(8):351–360.
2. Byrne B, Donohoe GG, O’Kennedy R. Sialic Acids: Carbohydrate Moieties that influence the biological and physical properties of biopharmaceutical Proteins and Living Cells. Drug Discov Today. 2007;12:319–326.
3. Padler-Karavani V, Yu H, Cao H, et al. Diversity in Specificity, Abundance, and Composition of Anti-Neu5Gc Antibodies in Normal Humans: Potential Implications for Disease. Glycobiology. 2008;18:818–830.
4. Thermo Fisher Scientific. Application Update 180: Direct Determination of Sialic Acids in Glycoprotein Hydrolyzates by HPAE-PAD. 2018.
5. Thermo Scientific Dionex ICS-6000 Ion Chromatography System Operator’s Manual. P/N 22181-97002. 2018.
6. Thermo Scientific. Electrochemical Detection User’s Compendium. P/N 065340-02. 2013.
7. Thermo Scientific Dionex ICS-6000 Ion Chromatography System Dual EGC Mode Installation. P/N 065760. 2020.
8. Thermo Fisher Scientific Technical Note 41: Analysis of Sialic Acids Using High-Performance Anion-Exchange Chromatography. 2000.
9. Thermo Scientific Dionex CarboPac PA20 Columns Product Manual. P/N 031884. 2018.
10. Thermo Scientific. Bio-IC Column Selection Guide. 2021.
11. Thermo Fisher Scientific. Application Update 181: Rapid Screening of Sialic Acids in Glycoproteins by HPAE-PAD.
12. Thermo Fisher Scientific. Application Note 253: HPAE-PAD Determination of Infant Formula Sialic Acids.
13. ICH Q2B Validation of Analytical Procedures: Methodology. CPMP/ICH/281/95. 1996.