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.
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