Amide-Bonded BEH HILIC Columns for High Resolution, HPLC-Compatible Separations of N-Glycans

Significance of the Topic

Glycosylation critically influences the safety, efficacy and stability of biotherapeutic proteins. Detailed profiling of N-glycans is essential in research and quality control to detect process variations and ensure consistent product performance. Hydrophilic interaction chromatography (HILIC) combined with fluorescence labeling has become a standard approach, but improvements in resolution, speed and method transferability between UPLC and HPLC systems remain in high demand.

Study Objectives and Overview

This work evaluates amide-bonded ethylene-bridged hybrid (BEH) HILIC columns packed with 1.7, 2.5 and 3.5 µm particles to:

• Compare resolution and peak capacity for 2-AB labeled N-glycans under UPLC-compatible and HPLC-compatible conditions
• Demonstrate method scaling between particle sizes to maintain selectivity
• Assess consistency of glycan profiles and glucose unit (GU) assignments across columns and instrumentation

Methodology and Instrumentation

All separations used fluorescently labeled N-glycans derived from human serum IgG and spiked A3 species. Gradient conditions were optimized for the 1.7 µm column and scaled for 2.5 and 3.5 µm phases by adjusting flow rate and gradient time according to particle diameter ratios. Peak capacities were calculated from half-height peak widths over the full elution window. Selectivity factors and GU values were assigned using a dextran calibration ladder.

Instrumentation

• UPLC: ACQUITY UPLC H-Class Bio System with FLR detectors (330 nm excitation, 420 nm emission)
• HPLC: Alliance HPLC System with 2475 FLR detector
• Columns: ACQUITY UPLC BEH Glycan (1.7 µm), XBridge BEH Glycan (2.5 µm XP and 3.5 µm), competitor amide-bonded silica (3 µm)
• Calibration Standards: 2-AB Labeled Glycan Performance Test Standard, Dextran Calibration Ladder
• Data Processing: Waters UNIFI and Empower Pro software

Main Results and Discussion

Under UPLC conditions at identical linear velocity, the 1.7 µm BEH Glycan column achieved the highest peak capacity (Pc=158), while the 2.5 µm and 3.5 µm phases delivered Pc=135 and 105 respectively, outperforming a 3 µm silica competitor (Pc=93). Column pressures decreased inversely with particle size, enabling HPLC compatibility for 2.5 µm (4,600 psi) and 3.5 µm (1,800 psi) phases. Scaled HPLC methods recovered up to 98% of UPLC peak capacity for the 2.5 µm column by extending gradient time and lowering flow rate. Selectivity factors remained consistent across columns, preserving elution order. Relative glycan abundances were within 10% agreement except for minor deviations in co-eluting species on the 3.5 µm phase. GU values differed by less than 0.05 units, well within typical database error.

Benefits and Practical Applications

• Flexible method transfer between UPLC and HPLC without altering glycan profiles or GU calibration
• Enhanced resolution and throughput options to match available instrumentation
• Robust batch-to-batch performance supporting QC in biopharma settings

Future Trends and Opportunities

Continued integration of sub-2 µm BEH phases with routine HPLC platforms can broaden access to high-resolution glycan analysis. Further exploration of UHPLC-MS coupling and automated GU libraries will streamline identification workflows. Advances in stationary phases targeting other glycan classes, such as O-linked and sialylated species, offer avenues for comprehensive glycoprotein profiling.

Conclusion

The BEH Glycan column family spanning 1.7 to 3.5 µm particle sizes delivers scalable, high-resolution separations of N-glycans across UPLC and HPLC systems. Method scaling preserves selectivity and GU accuracy, enabling flexible implementation in research and quality control laboratories.

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