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

Significance of the Topic

Glycosylation of therapeutic proteins is a critical quality attribute affecting function, stability and serum half-life.
Reliable profiling of released N-glycans ensures biotherapeutic safety and consistency.

Objectives and Study Overview

This study evaluates the performance of the Agilent AdvanceBio Amide HILIC column combined with Agilent AdvanceBio Ammonium Formate concentrate for LC/FLD/MS analysis of released N-glycans from monoclonal antibodies including NISTmAb, rituximab and cetuximab.
The goal was to assess selectivity, resolution and throughput compared with existing HILIC columns.

Methodology

N-glycans were enzymatically released from mAbs and labeled with InstantPC dye.
Labeled glycans were purified using the Agilent AdvanceBio Gly-X N-glycan prep kit.
Separations were performed under HILIC conditions using a gradient of ammonium formate buffer and acetonitrile.
Detection was achieved with fluorescence (λEx 285 nm, λEm 345 nm) and Q-TOF mass spectrometry.

Instrumentation

• Agilent 1290 Infinity II LC system with bio multicolumn thermostat, ultralow dispersion kit and FLD flow cell
• Agilent AdvanceBio Amide HILIC column (2.1 × 150 mm, 1.8 μm)
• Mobile phase: 50 mM ammonium formate (pH 4.4) and acetonitrile
• Agilent 6545XT AdvanceBio LC/Q-TOF with Dual AJS ESI source

Results and Discussion

Simple glycan mixtures from NISTmAb and rituximab showed baseline separation of major neutral species (G0F, Man5, G1[6]F etc.) in ~15 minutes with high peak shape quality.
Complex cetuximab glycans including sialylated and Galα1-3Gal/NeuGc epitopes were profiled in a 40-minute window, revealing numerous well-resolved peaks in the FLD trace and additional minor species in MS-extracted chromatograms.
MS/MS confirmation of FA2G2Ga2 structure demonstrated the column’s suitability for downstream structural analysis.
Head-to-head comparison with a Waters Premier BEH Amide column highlighted enhanced charge-group selectivity and reduced coelution on the Agilent column.

Benefits and Practical Applications

Improved charge-group selectivity minimizes coelution of critical glycan pairs.
Robust peak shape and retention stability support high-throughput QC and R&D workflows.
Use of ready-to-use ammonium formate concentrate reduces buffer preparation time and variability.

Future Trends and Applications

Integration with advanced MS/MS and software algorithms for automated glycan annotation.
Adoption in process analytical technology (PAT) for real-time glycosylation monitoring.
Extension to glycopeptide and intact glycoprotein analysis for deeper structural insights.
Development of multiplexed labeling strategies and retention time alignment across platforms.

Conclusion

The Agilent AdvanceBio Amide HILIC column with ammonium formate concentrate delivers fast, selective and reproducible separation of released N-glycans.
Its superior charge selectivity, compatibility with FLD and MS, and ease of use make it a valuable tool for biopharmaceutical glycan analysis.

References

1. Delobel, A. Glycosylation of Therapeutic Proteins: A Critical Quality Attribute. In Mass Spectrometry of Glycoproteins: Methods and Protocols; Springer US, 2021; pp 1–21.
2. Zhang, X.; Vimalraj, V.; Patel, M. Routine Analysis of N-Glycans Using Liquid Chromatography Coupled to Routine Mass Detection. In Mass Spectrometry of Glycoproteins: Methods and Protocols; Springer US, 2021; pp 205–219.