Enhancing the Peak Capacity of High Molecular Weight N-Glycan HILIC Separations with a Wide-Pore Amide Bonded Stationary Phase

Significance of the Topic

Reliable analysis of N-linked glycans is fundamental to the development and quality control of biopharmaceuticals. Complex glycan structures, especially high molecular weight tri- and tetra-antennary species, influence efficacy, immunogenicity, and stability of glycoprotein therapeutics. Optimizing chromatographic resolution for these large glycans is therefore crucial for detailed profiling and regulatory compliance.

Study Objectives and Overview

This study evaluates the performance of a wide-pore amide bonded stationary phase (300 Å, 1.7 µm) compared to a conventional glycan column (130 Å, 1.7 µm) for UPLC-HILIC separations of RapiFluor-MS labeled N-glycans. Two sample types were examined: biantennary glycans from pooled human IgG and highly branched glycans from recombinant coagulation Factor IX.

Methodology and Instrumentation

Sample Preparation

• Rapid deglycosylation and fluorescent/MS-active labeling using RapiFluor-MS reagent
• μElution HILIC solid phase extraction following GlycoWorks kit protocol

Chromatographic Conditions

• Separation by HILIC on ACQUITY UPLC columns (2.1×150 mm, 1.7 µm)
• Comparative stationary phases: glycan BEH amide 130 Å vs glycoprotein BEH amide 300 Å
• Mobile phase gradients and flow rates per GlycoWorks Care and Use Manual
• Fluorescence detection of labeled glycans

Instrumentation Used

• ACQUITY UPLC system
• Glycan BEH amide column, 130 Å, 1.7 µm, 2.1×150 mm
• Glycoprotein BEH amide column, 300 Å, 1.7 µm, 2.1×150 mm
• RapiFluor-MS N-Glycan Kit
• Fluorescence detector module

Results and Discussion

Chromatograms of IgG biantennary glycans showed similar peak capacities on both columns, indicating equivalent performance for smaller structures. In contrast, Factor IX tri- and tetra-antennary glycans exhibited a 17% increase in effective peak capacity on the 300 Å column (Pc 62 vs Pc 53). Peak widths at half height decreased from ~7.5 s to ~6.6 s, enhancing resolution of low-abundance species such as FA4G4 variants. Wide-pore pores allow large glycans unhindered access to the stationary phase network, reducing diffusional limitations and improving separation efficiency.

Benefits and Practical Applications

• Enhanced resolving power for highly branched high molecular weight glycans
• Improved detection of minor glycoforms critical for biosimilarity and lot release testing
• Facilitated profiling of complex biotherapeutics including Factor IX erythropoietin and darbepoetin
• Streamlined integration into existing UPLC-HILIC workflows

Future Trends and Applications

Further development may include coupling wide-pore HILIC separations with high-resolution mass spectrometry for intact glycoprotein analysis. Expansion of pore sizes and particle technologies could enhance performance for even larger biomolecules. Adoption in regulated labs will drive advances in automated glycan profiling and database matching.

Conclusion

The wide-pore (300 Å) BEH amide column offers a clear advantage for UPLC-HILIC separation of high molecular weight tri- and tetra-antennary N-glycans, delivering up to 17% greater peak capacity and improved resolution of low-abundance species. Conventional 130 Å columns remain suitable for smaller biantennary structures, while the 300 Å phase is recommended for detailed analysis of complex glycan profiles in biopharmaceutical development and quality control.

References

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