Glycoprofiling

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

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.

Endo F Multi-Kit
Endo F Multi-Kit
ORELA O-linked deglycosylation image
O-linked Glycan Release Kit
PNGase F image showing cleavage of biantennary, triantennary, high mannose and hybrid glycans
PNgase F

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.
CEX Cartridges image
CEX Cartridges for O-Glycans
Ludger EB10 cartridges image
EB10 Cartridges
Ludger EC50 cartridge photo
EC50 Cartridges
PBM Glycosidase Removal Plate Image
Glycosidase Removal Plate

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.

2-AB Glycan labeling kit image
2-AB Labeling Kit
2-AA Labeling Kit
2-AA Labeling Kit
Procainamide Labeling Kit
Procainamide Labeling Kit
permethylation kit image
Permethylation Kit

Labeling Cleanup

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 our vacuum manifold system.

S cartridges image
S Cartridges
Ludger T1 Cartridges Image
T1 Cartridges
Velocity manifold
Velocity Vacuum Manifold

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
C3 anion exchange column
Anion Exchange
Normal phase N1 column
Normal Phase
R1 4.6x150mm HPLC column
Reverse Phase

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.