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
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.
Glycan Capture and Cleanup
A number of technologies have been developed for the purification of glycans from proteins, salts, and detergents.
- LudgerClean EB10 and EC50 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.
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.
One of the most reliable methods for the cleanup of glycan samples after fluorescent labeling is the use of S-cartridges. These employ a hydrophilic membrane to separate the labeled glycans from non-glycan contaminants and are routinely used in the biopharma industry during glycoprofiling QC for lot release of FDA and EMA approved biopharmaceuticals.
The T1 cartridges are a more recently developed technology designed to be faster and amenable to high-throughput analysis. Total procedure complete in 45 minutes when using the Ludger Velocity vacuum manifold system.
HPLC Columns and Buffers for Glycoprofiling
We supply a full line of Ludger HPLC columns and buffers developed specifically for glycan analysis.
- C2 and C3 anion exchange columns and buffer – charge based analysis
- N1 and N2 normal phase columns and buffer – glycoprofiling by size based analysis
- R1, R2, and uR2 reverse phase columns and solvent – sialic acid and monosacchride analysis
Glycan and Glycoprotein Standards
QA-Bio also supplies a wide variety of qualitative and quantitative glycan standards and glycoprotein standards to be used during glycoprofiling. All are produced at the highest quality and purity levels.
- Glycans – Greater than 90% pure by NMR and HPLC
- Glycan Libraries – Fetuin, IgG
- Quantitative Standards – determined by qNMR and qMonosaccharide analysis
- Glycoprotein Standards – Fetuin, IgG