Released Glycan Analysis of Erythropoietin Using the ACQUITY Premier Glycan BEH C 18 AX Column and BioAccord System with ACQUITY Premier

Significance of the Topic

Glycosylation critically influences the pharmacokinetics, efficacy, and immunogenicity of biotherapeutics. Highly sialylated glycan structures, such as those on erythropoietin (EPO), affect clearance rates and in vivo activity. Accurate, high-resolution analytical methods are essential to monitor glycan heterogeneity and ensure consistent drug quality.

Study Objectives and Overview

This work evaluates a single-step mixed-mode chromatographic approach using the ACQUITY Premier Glycan BEH C18 AX Column combined with RapiFluor-MS labeling and the BioAccord LC-FLR-MS system. The goal is to enhance separation and detection of highly sialylated, complex N-glycans released from EPO, comparing performance against traditional HILIC methods.

Methodology

• Sample Preparation: N-glycans released from EPO reference materials and labeled with RapiFluor-MS tag following a quench step.
• Chromatography: Mixed-mode (IEX/RP) separation on C18 AX column versus HILIC on Glycan Amide column using ACQUITY Premier UPLC at 60 °C.
• Detection: Fluorescence detection (λ_ex=265 nm, λ_em=425 nm) and ESI⁺ MS (50–2000 m/z) with data-independent fragmentation.

Instrumentation

• BioAccord System with ACQUITY Premier UPLC.
• ACQUITY Premier Glycan BEH C18 AX Column (1.7 µm, 2.1×150 mm) and Glycan Amide HILIC Column.
• ACQUITY Premier FLR Detector and ACQUITY RDa Mass Detector.
• waters_connect informatics platform with UNIFI.

Key Results and Discussion

The mixed-mode C18 AX method achieved primary separation based on glycan charge (0–4 sialic acids) and secondary separation by hydrophobicity, yielding ~20 % narrower peaks versus HILIC. In EPO analyses, 82 unique glycan masses were detected from 1 µg protein. Isomeric species such as FA4S3 were resolved to near-baseline. DIA fragmentation spectra enabled clear structural assignments, exemplified by stepwise monosaccharide losses confirming the high-mannose M7P2 glycan.

Benefits and Practical Applications

• Enhanced chromatographic resolution for highly sialylated glycans compared to HILIC.
• Charge-based profiling simplifies interpretation of complex glycan mixtures.
• Improved FLR-MS sensitivity allows detection down to 0.1 % relative abundance with minimal sample input.
• Integrated DIA fragmentation supports confident structural elucidation.
• Single-step protocol reduces development time and per-sample cost versus two-stage fractionation.
• Applicable for comparability studies across biotherapeutic batches.

Future Trends and Potential Applications

• Extension to other highly sialylated biotherapeutics and glycoproteins.
• Automation of DIA data processing for high-throughput structural analysis.
• Adoption of MS-compatible mixed-mode phases for diverse biomolecule classes.
• Integration into quality control and bioprocess monitoring workflows.

Conclusion

The ACQUITY Premier Glycan BEH C18 AX mixed-mode column, combined with RapiFluor-MS labeling and the BioAccord LC-FLR-MS system, delivers superior resolution, sensitivity, and charge-based selectivity for complex, highly sialylated N-glycans. This streamlined approach enhances glycan profiling, structural assignment, and comparability assessment in biotherapeutic development and quality control.

References

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3. Udayanath A. et al. Separation of 2AB Labeled N-glycans from Bovine Fetuin on a Novel Mixed-Mode Stationary Phase. Thermo Fisher Scientific Application Note 2075A.
4. Liu X. et al. Increased Resolving Power for Acidic Glycans with an MS-Compatible Anion Exchange Reversed-Phase Separation. Waters Application Note 720007038. 2020.