Glycosaminoglycans

• The CCRC Analytical Services group is well trained in the complete isolation and purification of GAG from cells and tissue.
• Once GAGs are isolated they can be purified or analyzed in-house by methods including disaccharide composition analysis, ion-exchange chromatography, size exclusion chromatography, MS and NMR.

• While highly diverse when whole, the use of highly specific enzymes may be used to break down a GAG structure into its component disaccharides, giving valuable information regarding its composition.
• We routinely perform GAG disaccharide analysis using either NSI-MS/MS or on one of the Agilent 1200 HPLC instruments in house. These instruments may be coupled to UV-VIS or in case of HPLC for fluorescence detection, as part of a post-column labeling instrument that provides limits of detection as low as 1-10ng of injected disaccharide (for fluorescence).
• Alternatively, GAG disaccharides may be separated with Capillary Electrophoresis. The CCRC is equipped with a Beckman Coulter PA800 Pharmaceutical Capillary Electrophoresis system, as well as a CESI 8000 – a nanospray-compatible instrument for coupling with mass spectrometry.

Services being offered for glycosaminoglycan (GAG) structural analysis:

• Enzyme digestion of glycosaminoglycans to disaccharides
• Chemical degradation and HPLC, MS or NMR analysis
• Oligosaccharide profiling by CTA-SAX-HPLC
• Identification of disaccharides and sulfation pattern by online ESI-MS and ESI-MS/MS
• Identification of released disaccharides by Strong-Anion-Exchange Chromatography, and detection of the disaccharides by both UV and fluorescent detection.
• Structural characterization of GAG oligosaccharides by 1D and 2D NMR
• Sequencing of GAG oligosaccharides by ESI-MS and ESI-MS/MS
• Glycosyl composition analysis of GAGs by GC-MS

• Mass spectrometry: MS technique provides detection and identification of disaccharides and sulfation patterns; sequencing of GAG oligosaccharides; detection and identification of the oligosaccharides terminating with 1,6-anhydro GlcNH2, 1,6-anhydro ManNH2 or 2,5-anhydro mannitol.
• One-dimensional NMR: Proton and carbon spectra provide overall chemical composition of polysaccharides and spectroscopic fingerprints of GAG oligosaccharides.
• Two-dimensional NMR: HSQC spectrum provides the ratio information of glucosamine to mannosamine (A/M) as well as the ratio of iduronic acid to glucuronic acid (I/G) of GAGs, such as heparin sodium, desulfated heparin sodium and LMWHs.

We offer method development, method validation and structural characterization study of many different sulfated products including low-molecular-weight heparins (LMWH) to evaluate active ingredient sameness of the generic and innovator products.

List of our services:

• Equivalence of physicochemical properties: This includes determination of molecular weight distribution by SEC-HPLC; chain mapping by CTA-SAX and LC-MS; presence of common functional groups by proton and carbon NMR spectroscopy and molar ratio of sulfate to carboxylate by conductivity titration.
• Equivalence of source material and mode of depolymerization: This study includes monosaccharide analysis by HPAEC; proton NMR and disaccharide analysis of the product.

Equivalence of disaccharide building blocks, fragment mapping and sequence of oligosaccharide species:

This study includes disaccharide building blocks analysis using exhaustive digestion by SAX-HPLC and LC-MS; fragment mapping individual enzymes; sequence of oligosaccharides by separation and 2D NMR; determination of ratio of modified sugars by 2D NMR spectroscopy.