This module provides WAX-HPLC fractionization, followed by exoglycosidase sequencing of each collected pool.
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 control, and alongside Ludger’s system suitability standards, positive and negative controls.
This module is suitable for:
- sialylated samples
- quality control – detailed profile comparisons to monitor charge distribution
- monitoring batch to batch consistency
- comparability studies
Workflow for Level 1 WAX 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 labeled with 2-AB purified and analysed on a LudgerSep-C3 weak anion exchange (WAX) column. Fractions are pooled and characterized by exoglycosidase sequencing.
WAX-HPLC provides charge profiles for profile comparison between batches where glycan separation is based on glycan charge.
Final report contains:
- WAX-HPLC profiles for system suitability standards and Ludger positive controls
- WAX-HPLC profiles for client samples and buffer negative controls
- Relative proportions of the mono-, di-, tri- and tetra-sialylated glycan structures
- HILIC-UPLC profiles for system suitability standards and Ludger positive controls before and after exoglycosidase digestions
- HILIC-UPLC profiles for client samples and buffer negative controls before and after exoglycosidase digestions
- Glucose unit (GU) values
- Glycan structures (including monosaccharide type, order and linkage) and their relative proportions
- Summary of findings (e.g. G0:G1:G2; % high mannose; % fucosylation; % Galα1-3Gal)