Improved Hydrophilic Interaction Liquid Chromatography for LC/MS Analysis of Released N-Glycans

Importance of the Topic

Glycosylation is a critical post-translational modification that influences protein folding, stability and biological activity. In biotherapeutic development, accurate characterization of N-linked glycans is essential for ensuring product quality, safety and efficacy. Hydrophilic interaction liquid chromatography (HILIC) coupled with mass spectrometry (MS) has become a standard approach for detailed glycan profiling, but challenges remain in resolving closely related glycan species and preventing undesirable interactions with metal surfaces in the LC flow path.

Objectives and Study Overview

This study aimed to develop and evaluate a novel amide-based HILIC stationary phase that:

• Allows modulation of glycan charge group selectivity via mobile phase buffer strength within an MS-compatible range
• Enables rapid separation of complex N-glycan mixtures in a shortened gradient window
• Incorporates a low-concentration deactivator additive to suppress metal-glycan interactions on standard steel systems

Methodology and Instrumentation

Released N-glycans from therapeutic antibodies were labeled with InstantPC and analyzed using two LC-MS platforms:

• Agilent 6545XT AdvanceBio LC/Q-TOF with 1290 Infinity II Bio LC: prototype amide HILIC column (2.1 × 150 mm, 1.8 μm) at 60 °C; mobile phases A: 50 mM ammonium formate (pH 4.4), B: acetonitrile; flow 0.6 mL/min; gradient 77–48% B over 45 min.
• Agilent 6230 LC/TOF with 1290 Infinity II LC: same column and temperature; mobile phase A: 50 mM ammonium formate (pH 4.4) plus 5 μM InfinityLab Deactivator Additive, B: acetonitrile; flow 0.5 mL/min; gradient 77–42.5% B over 36 min.

Main Results and Discussion

Key findings include:

• Charge group selectivity could be tuned by varying ammonium formate concentration from 50 to 100 mM, improving resolution of critical glycan pairs without excessively long gradients.
• The prototype column achieved high peak capacity for complex glycan profiles (e.g., cetuximab), resolving neutral, mono- and multi-sialylated structures within a 30-min window.
• Incorporation of 5 μM deactivator additive prevented peak broadening of acidic glycans on a steel LC system, demonstrating effective metal site passivation while maintaining MS sensitivity.

Practical Benefits and Applications

This optimized HILIC-MS workflow offers:

• Enhanced selectivity for detailed glycan profiling in biopharma QA/QC laboratories.
• Reduced analysis time for high-complexity samples, facilitating higher throughput.
• Compatibility with standard LC hardware through simple additive use to suppress metal interactions.

Future Trends and Potential Applications

Anticipated developments include:

• Further customization of stationary phase chemistries for isomeric glycan separations.
• Integration into automated, high-throughput glycomics platforms.
• Extension of the approach to other glycoprotein classes and glycan labeling strategies.
• Wider adoption of MS-compatible deactivator additives in routine LC-MS analyses.

Conclusion

The novel amide HILIC stationary phase combined with an MS-compatible deactivator additive enables flexible charge-based selectivity and effective mitigation of metal-glycan interactions. This approach delivers rapid, high-resolution glycan profiling suitable for both research and regulated environments.

References

• Yang X., Bartlett M. J. Chromatogr. B. 2019, 1120, 29–40.
• Reusch D., Tejada M. L. Glycobiology. 2015, 25, 1325–1334.