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:

• Release of glycans from glycoprotein
• capture and cleanup of released glycans
• labeling of released glycans
• purification and cleanup of labeled glycans
• 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. 

QAbio  Glycan Capture and Cleanup

A number of technologies have been developed for the purification of glycans from proteins, salts, and detergents.

• LudgerClean EB10 cartridges have been designed for purification of glycans from proteins, salts, and detergents.
• The LudgerClean PBM Post-Glycosidase Clean-Up plate has been developed for removal of glycosidases prior to MS analysis in a 96-well format.
• LudgerClean CEX cartridges purify O-glycans from non-carbohydrate material including salts, proteins, and detergents.

QAbio  Glycan Labeling

Glycans are then typically labeled with a reporter molecule based upon the separation/detection systems being used for the analysis. Ludger has developed a variety of robust and reliable glycan labeling kits.

Fluorescent molecules 2-AB and 2-AA have been used for glycan analysis in HPLC assays for many years and are considered a gold standard within the industry.

Permethylation labeling kits are available for mass spectrometry and APTS have been developed for capillary electrophoresis.

Procainamide labelling permits glycan identification by either mass spectrometry or UHPLC, and because of its improved ionisation efficiency it can permit identification of minor glycans (>1% relative peak area) by ESI-MS.