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Glycan Mapping Workflow

A typical workflow for glycan analysis of glycosylated protein biopharmaceuticals is shown in Figure 1 and described below.

Typical Glycan Analysis Workflow

Figure 1: Typical glycan analysis workflow

Typical Glycan Analysis Workflow
Figure 2: 2-AB labeling reaction
  1. 50 µg of the protein biopharmaceutical is reduced with 5 mM dithiotreitol and alkylated with 10 mM iodoacetamide in ammonium bicarbonate at pH 8
  2.  
    1. The glycans are then liberated by adding 0.1 – 0.2 units of PNGase F per µg of the protein biopharmaceutical.  The sample is then incubated at 37 °C for 18 hours
    2. Excess PNGase F and protein biopharmaceutical are then removed by a HILIC SPE clean-up step – the sample is loaded in 90% acetonitrile (aq) and eluted in 1 mM ammonium acetate in 10% acetonitrile.
  3.  
    1. Following drying, the glycans are labelled with 2-aminobenzamide (2-AB) via reductive amination in the presence of sodium cyanoborohydride and then incubated for 3 hours at 65 °C
  4. The dried glycans are then dissolved in 25 µL water, vortex mixed and diluted with an equal portion of acetonitrile

The sample is then ready for hydrpophilic interaction iquid chromatography (HILIC) analysis.

 

 

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