Structural characterization of heparins from different commercial sources

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 electrop...

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Bibliographic Details
Published in:	Analytical and bioanalytical chemistry Vol. 401; no. 9; pp. 2793 - 2803
Main Authors:	Zhang, Fuming, Yang, Bo, Ly, Mellisa, Solakyildirim, Kemal, Xiao, Zhongping, Wang, Zhenyu, Beaudet, Julie M., Torelli, Amanda Y., Dordick, Jonathan S., Linhardt, Robert J.
Format:	Journal Article
Language:	English
Published:	Berlin/Heidelberg Springer-Verlag 01-11-2011 Springer
Subjects:	Analytical Chemistry Animals Antithrombin III - metabolism Binding Biochemistry Bioengineering Carbohydrate Sequence Characterization and Evaluation of Materials Chemical properties Chemistry Chemistry and Materials Science Chemistry, Physical - methods Chromatographic methods and physical methods associated with chromatography Chromatography, Gel Chromatography, High Pressure Liquid - methods Electrophoresis Electrophoresis, Polyacrylamide Gel Exact sciences and technology Food Science Glucosamine - analysis Heparin Heparin - analysis Heparin - chemistry Heparin Lyase - metabolism Heparins High performance liquid chromatography Laboratory Medicine Magnetic resonance Magnetic Resonance Spectroscopy - methods Molecular Sequence Data Molecular Weight Monitoring/Environmental Analysis Nuclear magnetic resonance Nuclear magnetic resonance spectroscopy Original Paper Other chromatographic methods Protein Binding Spectrometric and optical methods Surface Plasmon Resonance - methods Swine Thrombin Thrombin - metabolism Polyacrylamide gel electrophoresis Disaccharide composition Oligosaccharide mapping Size exclusion chromatography analysis High performance liquid chromatography–mass spectrometry Nuclear magnetic resonance spectroscopy Surface plasmon resonance Heparin Molecular weight properties Glucosamine High performance liquid chromatography-mass spectrometry Chemical analysis HPLC chromatography NMR spectrometry Disaccharide Characterization Design Thrombin(drug) Peak Molecular mass Iodine III Gel electrophoresis Sample Oligosaccharide Spectral analysis Gel permeation chromatography Treatment Residue Acrylamide polymer Affinity Mass spectrometry Physicochemical properties
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Summary:	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 1 H- and 13 C-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.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	1618-2642 1618-2650
DOI:	10.1007/s00216-011-5367-7