title: Structural characterization of heparins from different commercial sources.
abstract: 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.
authors.affiliation: Department of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA. zhangf2@rpi.edu
authors.forename: Fuming
authors.initials: F
authors.lastname: Zhang
authors.forename: Bo
authors.initials: B
authors.lastname: Yang
authors.forename: Mellisa
authors.initials: M
authors.lastname: Ly
authors.forename: Kemal
authors.initials: K
authors.lastname: Solakyildirim
authors.forename: Zhongping
authors.initials: Z
authors.lastname: Xiao
authors.forename: Zhenyu
authors.initials: Z
authors.lastname: Wang
authors.forename: Julie M
authors.initials: JM
authors.lastname: Beaudet
authors.forename: Amanda Y
authors.initials: AY
authors.lastname: Torelli
authors.forename: Jonathan S
authors.initials: JS
authors.lastname: Dordick
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Linhardt
mesh_terms: D000818:Animals; D000990:Antithrombin III / Q000378:metabolism; D002240:Carbohydrate Sequence; D002627:Chemistry, Physical / Q000379:methods*; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D005944:Glucosamine / Q000032:analysis; D006493:Heparin / Q000032:analysis* / Q000737:chemistry; D019763:Heparin Lyase / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011485:Protein Binding; D020349:Surface Plasmon Resonance / Q000379:methods*; D013552:Swine; D013917:Thrombin / Q000378:metabolism
chemical_list: D000990:Antithrombin III; D006493:Heparin; D013917:Thrombin; D019763:Heparin Lyase; D005944:Glucosamine