HILIC Profiling

HILIC glycan analysis image

This module provides HILIC-UHPLC N- and/or O-glycan profiles with relative quantitation of peaks and preliminary identification of the main glycan species. The analysis will be performed on single or triplicate releases of samples (typically 5-100 µg for each release), an equivalent amount (by volume) of sample buffer negative controls, alongside Ludger positive and negative controls and system suitability standards.

This module is suitable for:

  • preliminary glycan assignment
  • quality control – profile comparisons to monitor known structures
  • monitoring batch to batch consistency
  • comparability studies

In order to gain more detailed information on the glycan structures and their relative proportions Level 2 Detailed Characterization is required.

Workflow for Level 1 HILIC profiling

N-glycans are released from glycoprotein by digestion with PNGAse F or PNGAse A.
O-glycans are released from glycoprotein by hydrazinolysis or Orela reagent.
Released glycans are fluorescently labelled with 2-AB or 2-AA or procainamide, purified and analysed by HILIC-HPLC or HILIC-UHPLC.

HILIC-HPLC/UHPLC provides glucose unit (GU) values for profiles where glycan separation is roughly based on glycan size and allows direct profile comparison between samples and standards.

Report

Final report contains:

  • HILIC-UHPLC profiles for system suitability standards and Ludger positive controls
  • HILIC-UHPLC profiles for client samples and buffer negative controls
  • Glucose unit (GU) values
  • Relative proportions of detected peaks
  • Summary of findings

Comparison of the glycans in client samples with Ludger’s glycan standards allows for preliminary identification of the main glycan species

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