Developing High Resolution HILIC Separations of Intact Glycosylated Proteins Using a Wide-Pore Amide-Bonded Stationary Phase

Significance of the topic

Glycosylation is one of the most important post-translational modifications affecting protein stability, pharmacokinetics and immunogenicity.
Accurate separation and profiling of intact glycoproteins and their glycoforms are therefore essential for biopharmaceutical development, quality control, and regulatory compliance.
Hydrophilic interaction liquid chromatography (HILIC) holds promise for resolving highly polar glycoforms but has been underutilized for large biomolecules due to challenges such as protein precipitation and pore accessibility.

Objectives and study overview

This work presents the design and application of a wide-pore, amide-bonded BEH stationary phase (300 Å, 1.7 µm) for high-resolution HILIC of intact glycosylated proteins.
The goals were to:

• Optimize stationary-phase pore size, ion-pairing agents and injection conditions for intact proteins.
• Benchmark performance against existing HILIC and reversed-phase materials using model glycoproteins (RNase A/B, trastuzumab, Intact mAb test standards).
• Demonstrate MS-compatibility and orthogonality to reversed-phase separations.
• Develop assays for glycan occupancy and heterogeneity of intact monoclonal antibodies.

Methodology and instrumentation

Key methodological features included:

• Stationary phase: ACQUITY UPLC Glycoprotein BEH Amide, 300 Å, 1.7 µm, ethylene-bridged hybrid particles.
• Chromatography: ACQUITY UPLC H-Class Bio System operated at 0.2 mL/min, 30–45 °C, with focus on minimizing protein precipitation by limiting aqueous injection volumes to ≤ 1 µL on 2.1 mm I.D. columns.
• Mobile phases: 0.1% TFA in water (A) and 0.1% TFA in ACN (B) for optimal glycoform resolution; ammonium formate and formic acid were evaluated but found inferior.
• MS detection: Xevo G2 QTof, ESI+ mode, desolvation at 350 °C, scan 500–4000 m/z; MassLynx® for data processing.
• Column coupling: Use of low-volume ultrahigh-pressure PEEK connectors and optional post-column restriction tubing to raise operating pressures up to ~7,500 psi for intact IgGs.

Main results and discussion

Progressive improvements in resolving RNase B glycoforms were observed when moving from unbonded HILIC (130 Å, 5 µm) to amide-bonded BEH (130 Å, 1.7 µm) and ultimately to wide-pore BEH Amide (300 Å, 1.7 µm).
Wide-pore amide material delivered ~24% higher resolution between aglycosylated RNase A and Man5-RNase B and baseline separation of Man5–Man9 glycoforms.
Among 10 commercial HILIC phases tested, only amide-bonded materials with ≥ 100 Å pores achieved acceptable retention and resolution of RNase B.
Ion pairing with TFA at 0.1% improved retention selectivity, protonating acidic and basic residues to focus retention on glycan moieties and enabling on-line ESI-MS profiling.
HILIC-MS confirmed the identity of each RNase B glycoform (Man5–Man9) and the aglycosylated species via deconvoluted mass spectra.
Reversed-phase HPLC (C4) separated aglycosylated from glycosylated RNase B but could not resolve individual high-mannose glycoforms, demonstrating the orthogonal selectivity of HILIC.
Intact trastuzumab exhibited multiple glycoforms (G0F/G0F to G2F/G2F) on a single 150 mm BEH Amide column; at standard pressures peaks tailing and on-column aggregation occurred.
Coupling two 150 mm columns with a post-column restriction increased pressure (~7,000 psi), eliminated aggregation artifacts and revealed five major intact IgG glycoforms by UV and extracted ion chromatograms.
A stability study of PNGase F-treated Intact mAb Mass Check Standard at 80 °C using HFIP/TFA mobile phase allowed separation of native, partially and fully deglycosylated species, facilitating rapid glycan occupancy assays.

Benefits and practical applications

High-resolution HILIC of intact glycoproteins offers:

• Enhanced characterization of glycoform heterogeneity without glycan release.
• MS-compatible protocols for detailed mass mapping of intact species.
• Orthogonal data to reversed-phase assays for comprehensive QC workflows.
• Rapid glycan occupancy assays enabling determination of site occupancy and enzyme efficiency.
• Batch-to-batch reproducibility supported by QC testing on BEH Amide phases.

Future trends and potential applications

Continued integration of HILIC with high-resolution MS will enable automated intact glycoprotein profiling in biopharma pipelines.
Further miniaturization and ultrahigh-pressure UPLC may support higher throughput and stronger resolving power for larger protein complexes.
Adoption of ion-pairing strategies and novel co-solvents (HFIP, DMSO) may expand the range of proteins amenable to HILIC analysis.
Potential extensions include top-down glycoproteomics, site-specific glycoform mapping, and biosimilar comparability studies.

Conclusion

The development of a wide-pore (300 Å), amide-bonded BEH stationary phase has unlocked high-resolution HILIC separations of intact glycoproteins.
Optimization of mobile phases, column pressure and injection conditions provided baseline separation of complex glycoform mixtures and enabled MS characterization.
This methodology offers a powerful, orthogonal tool for glycoprotein analysis in research and QC environments.

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