Label-Free Analysis by HPLC with Charged Aerosol Detection of Glycans Separated by Charge, Size and Isomeric Structure

Significance of the Topic

Glycosylation of proteins is a fundamental post-translational modification that influences protein stability, activity, immunogenicity and biological recognition. Native N-glycans lack strong chromophores, so conventional methods require fluorescent or MS-compatible labeling to detect and quantify them. A label-free approach using high-performance separation combined with charged aerosol detection (CAD) offers a universal, sensitive solution for direct glycan profiling, supporting research, quality control and biopharmaceutical development.

Objectives and Study Overview

This study aimed to develop and validate a rapid, robust UHPLC-CAD assay for quantifying native N-linked glycans released from glycoproteins. Key goals included eliminating derivatization steps, achieving high chromatographic resolution of glycan isomers and charge variants, and demonstrating quantitative performance on standard glycoproteins (bovine fetuin, α1-acid glycoprotein).

Methodology and Instrumentation

Glycan release and sample preparation:

• PNGase F digestion (QA-Bio) on denatured glycoproteins;
• Centrifugation and direct injection of the released native glycans.

Chromatography and detection:

• Thermo Scientific Vanquish UHPLC system;
• GlycanPac AXR-1 mixed-mode column (weak anion exchange and reversed phase, 2.1×100 mm, 1.9 µm);
• Charged Aerosol Detector H with 50 °C evaporation temperature and 10 Hz data rate;
• Mobile phases: water and 100 mM ammonium formate pH 4.4;
• Gradient: linear increase from 4% to 39% buffer B at 1 mM/min.

Data acquisition and processing:

• Dionex Chromeleon CDS 7.2;
• Calibration with mono-, di- and tri-sialylated glycan standards (Prozyme) over 1–200 ng/µL.

Key Results and Discussion

The optimized UHPLC-CAD method achieved:

• High resolution separation of neutral, mono-, di-, tri- and tetra-sialylated glycan species by charge and size;
• Retention time precision better than 0.1% RSD;
• Peak area precision averaging 2–3% RSD across sialylated standards;
• Detection limits below 10 ng/µL (low picomole on-column);
• Linear dynamic range spanning two orders of magnitude with R²>0.995 (quadratic fit);
• No interference from PNGase F reagents or denaturants, with well-resolved glycan peaks.

Comparison of mobile phase composition, gradient slope and CAD evaporation temperature identified 5% organic modifier, 1 mM/min gradient slope and 50 °C as optimal conditions balancing sensitivity and run time.

Benefits and Practical Applications

• Eliminates time-consuming glycan labeling, reducing sample prep steps;
• Universal detection of any nonvolatile glycan, supporting diverse glycoprotein analyses;
• High throughput capability for QC labs to monitor lot-to-lot glycan profiles, degradation or impurity levels;
• Compatibility with MS-based workflows using a volatile mobile phase;
• Uniform aerosol response enables relative quantitation without pure standards for every glycan species.

Future Trends and Potential Applications

• Integration with high-resolution mass spectrometry for structural elucidation of glycan isomers;
• Automation of sample digestion and injection to support large-scale glycomics studies;
• Extension to other classes of glycans (O-glycans, glycolipids) and complex biotherapeutics;
• Development of specialized mixed-mode columns to further enhance isomer separation;
• Adoption in regulated environments for biopharmaceutical release testing and stability studies.

Conclusion

The UHPLC-CAD method provides a fast, label-free platform for sensitive, precise quantitation of native N-linked glycans. With minimal sample handling, robust performance and universal detection, it is well suited for research, industrial QC and integrated LC-MS glycan workflows.

References

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