Structural characterization of heparins from different commercial sources
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Seven commercial heparin active pharmaceutical ingredients and one commercial low molecular weight from different manufacturers were characterized with a view profiling their physicochemical properties. All heparins had similar molecular weight properties as determined by polyacrylamide gel electrophoresis (M N, 10–11 kDa; M W, 13–14 kDa; polydispersity (PD), 1.3–1.4) and by size exclusion chromatography (M N, 14–16 kDa; M W, 21–25 kDa; PD, 1.4–1.6). one-dimensional 1H- and 13C-nuclear magnetic resonance (NMR) evaluation of the heparin samples was performed, and peaks were fully assigned using two-dimensional NMR. The percentage of glucosamine residues with 3-O-sulfo groups and the percentage of N-sulfo groups and N-acetyl groups ranged from 5.8–7.9%, 78–82%, to 13–14%, respectively. There was substantial variability observed in the disaccharide composition, as determined by high performance liquid chromatography (HPLC)-mass spectral analysis of heparin lyase I–III digested heparins. Heparin oligosaccharide mapping was performed using HPLC following separate treatments with heparin lyase I, II, and III. These maps were useful in qualitatively and quantitatively identifying structural differences between these heparins. The binding affinities of these heparins to antithrombin III and thrombin were evaluated by using a surface plasmon resonance competitive binding assay. This study provides the physicochemical and activity characterization necessary for the appropriate design and synthesis of a generic bioengineered heparin.
KeywordsHeparin Polyacrylamide gel electrophoresis Size exclusion chromatography analysis Molecular weight properties Disaccharide composition High performance liquid chromatography–mass spectrometry Oligosaccharide mapping Nuclear magnetic resonance spectroscopy Surface plasmon resonance
This work was supported by grants funded by the National Institutes of Health HL101721 and HL096972 (RJL) and the Bioengineered Heparin Consortium.
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