Molecular Mass Characterization of Glycosaminoglycans with Different Degrees of Sulfation in Bioengineered Heparin Process by Size Exclusion Chromatography

Abstract

Different degrees of glycosaminoglycan sulfation result in their different charge densities. The charge density differences impact their migration behavior in polyacrylamide gel electrophoresis and size exclusion chromatography, two of the most common methods for determining relative molecular masses of polysaccharides. In this study, we investigated the feasibility of using commercially available heparin oligosaccharides as calibrants for measuring the relative molecular masses of intermediates in a bioengineered heparin process that have different levels of sulfation. A size exclusion chromatography method was established that eliminates this charge density effect and allows the determination of relative molecular mass using a single calibration curve with heparin oligosaccharides calibrants. This is accomplished by overcoming the electrostatic interaction between the glycosaminoglycans and size exclusion chromatography stationary phase using high ionic strength mobile phase.

Molecular weight and concentration of heparin in hyaluronic acid-based matrices modulates growth factor retention kinetics and stem cell fate

“…Although the aforementioned studies clearly demonstrated the value of heparin for growth factor delivery from bioinspired matrices, the attributes of heparin such as the extent of sulfation, pattern of sulfation, molecular weight, and polydispersity of heparin have been less emphasized and not explored in depth [29–32]. For example, heparin derivatives with different sulfation patterns differ significantly in their specific binding to VEGF 165 , which affects the subsequent bioactivity of VEGF 165 [24], and desulfated heparin-derivatives lose their stabilizing features and stimulatory capacity [33, 34].…”

Addressing endotoxin issues in bioengineered heparin

“…The resulting heparosan was >95% pure as determined by nuclear magnetic resonance (NMR) spectroscopy and disaccharide analysis ,. The average molecular weight determined by size‐exclusion chromatography (SEC) was ∼40,000 Da . The purified heparosan was freeze‐dried and stored desiccated at −20°C.…”

“…The purity of the N ‐acetyl, N ‐sulfoheparosan was determined by NMR spectroscopy and disaccharide analysis , as >95% with 12%–18% N ‐acetyl and 88%–82% N ‐sulfo content. The average molecular weight determined by SEC was 11,000–12,300 Da .…”

Is the endogenous ligand for PEAR1 a proteoglycan: clues from the sea

“…Heparin is the human glycosaminoglycan with the highest degree of sulfation, and one of the few glycosaminoglycans that can contain two or more sulfates on a single monomer. It also has a unique 3-O sulfation which is essential for its strong interaction with antithrombin III [30,31]. Most of the other glycosaminoglycans only have one sulfate group per monomer [31][32][33][34][35].…”

“…It also has a unique 3-O sulfation which is essential for its strong interaction with antithrombin III [30,31]. Most of the other glycosaminoglycans only have one sulfate group per monomer [31][32][33][34][35]. Heparan sulfate, one of the most abundantly expressed glycosaminoglycans that coats virtually every cell surface, has <1 one sulfate per disaccharide unit and does not contain the 3-0 sulfation that is unique for heparin [30,33,34].…”