HILIC – an alternative separation technique for glycopeptides

Significance of the topic

Glycosylation is a critical quality attribute of monoclonal antibodies (mAbs), influencing efficacy and safety. Detailed mapping of glycopeptides at the peptide level informs process control, stability assessment and regulatory compliance. Hydrophilic interaction liquid chromatography (HILIC) provides enhanced retention and resolution of glycopeptides compared to reversed-phase methods, enabling clear separation and quantitation.

Objectives and overview of the study

This study evaluates the performance of the Thermo Scientific Vanquish Flex UHPLC system coupled with an Accucore 150 Amide HILIC column for the simultaneous separation of peptides and glycopeptides derived from infliximab. Key aims included establishing retention time precision, sensitivity of UV versus fluorescence detection and the feasibility of accelerated gradient methods for routine analysis.

Methodology and instrumentation

Sample preparation involved digestion of infliximab (10 mg/mL) with the SMART Digest kit at 70°C for 45 minutes, reduction with DTT and cleanup on C18 spin columns. Peptides were eluted in 80% acetonitrile for HILIC loading. Chromatographic separation employed:

• Column: Accucore Amide HILIC, 2.1×150 mm, 2.6 μm, 150 Å
• Mobile phases: A – 90% acetonitrile/10% water; B – 10 mM ammonium formate pH 4.4 in water
• Gradient: full 10–60% B over 45 min; short 25–35% B over 1–15 min
• Flow rate: 0.5 mL/min; temperature: 50°C; injection volume: 24–50 μL
• Detectors: UV at 280 nm; fluorescence ex 280 nm/em 304 nm

Instrumental system: Vanquish Flex Quaternary UHPLC with SmartInject and Q Exactive HF Hybrid Quadrupole-Orbitrap MS for full MS and HCD MS2 acquisition.

Main results and discussion

The full gradient achieved baseline separation of infliximab glycopeptides, including isomers A2G1Fa and A2G1Fb, with retention time RSDs below 0.05%. A shortened gradient focused on the glycopeptide region (7–17 min) reduced analysis time and solvent usage while preserving resolution. MS1 and MS2 data permitted identification of 18 distinct glycoforms. Fluorescence detection yielded higher signal-to-noise and homogeneous response across glycopeptides compared to UV, enhancing quantitation of low-abundance species.

Benefits and practical applications

• Robust separation of glycopeptides versus nonglycosylated peptides for mAb characterization and QA/QC.
• High retention time precision supports confident peak assignment and method transfer to UV/FLD-based stability studies.
• Short gradient option increases throughput for routine monitoring of key glycoforms.
• FLD offers improved sensitivity for peptides containing tyrosine or tryptophan, enabling more comprehensive quantitation.

Future trends and opportunities

• Extension to middle-down and O-glycan profiling for more complex glycoproteins.
• Integration of automated data analysis and machine-learning for rapid glycoform interpretation.
• Development of high-throughput HILIC platforms for large-scale biopharma screening.
• Hybrid workflows combining released glycan labeling and glycopeptide mapping for complete glycosylation coverage.

Conclusion

The Vanquish Flex UHPLC system with Accucore 150 Amide HILIC column provides a reliable, precise and sensitive platform for glycopeptide analysis. Coupling UV or FLD detection with Orbitrap MS facilitates thorough characterization and efficient monitoring of mAb glycosylation profiles, supporting biotherapeutic development and quality control.

References

1. Thermo Scientific Application Note 1123. Increased Long-term Stability of Peptide Mapping using the Vanquish UHPLC System. Germering, Germany, 2015.
2. European Medicines Agency. EMA recommends approval of first two monoclonal-antibody biosimilars. 2013.
3. ICH Q6B Guideline for biotechnological products characterization. 1999.
4. EMA Guideline on similar biological medicinal products. 2014.
5. Thermo Scientific Application Note AN1132. Reliable results in Peptide Mapping using the Vanquish Flex UHPLC system. 2017.
6. Thermo Scientific Application Note AN1134. LC-UV-MS Vanquish Flex Peptide Mapping Development for transfer to LC-UV QA/QC. 2017.