Glycoprofiling is used by the biopharmaceutical industry to analyze the glycans found on glycoproteins being developed as therapeutics. It is also a key assay for the lot release characterization of manufactured glycoproteins both before and after approval.
The process can be broken down as follows:
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Release of glycans from glycoprotein
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capture and cleanup of released glycans
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labeling of released glycans
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purification and cleanup of labeled glycans
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analysis of glycan using MS, HPLC, or CE to produce the glycan profile
QAbio Glycan Release
Typically, PNGase F is used to release N-linked glycans. The enzyme works quickly and effectively, cleaving the glycans in native conditions in most cases. The Endo F family of enzymes are also commonly used for glycan release. These three enzymes vary in specificity to the multi-antennary structures found on N-linked glycans. They also differ from PNGase F in that they leave the penultimate glycan, a charged N-acetyl-glucosamine, attached to the protein. Both PNGase F and the Endo F family of enzymes leave the glycan with a free reducing terminii suitable for fluorescent labelling.
For O-linked glycan release, the Ludger Liberate Orela Kit allows the release of glycans that have free reducing terminii suitable for fluorescent labelling. The kit is straightforward and contains a simple method with no special handling techniques as required by the alternative method, hydrazinolysis.