WAX-Exoglycosidase Analysis

wax glycan analysis2 image

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.

Report

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)
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