Department of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA.
Anal Bioanal Chem. 2011 Nov;401(9):2793-803. doi: 10.1007/s00216-011-5367-7. Epub 2011 Sep 20.
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.
七种商业来源的肝素原料药和一种商业来源的低分子肝素，来自不同的制造商，其理化性质特征已被鉴定。所有肝素的重均分子量（M(W)）和数均分子量（M(N)）均相似，通过聚丙烯酰胺凝胶电泳（M(N)，10-11 kDa；M(W)，13-14 kDa；多分散系数（PD），1.3-1.4）和凝胶排阻色谱（M(N)，14-16 kDa；M(W)，21-25 kDa；PD，1.4-1.6）确定。采用一维（1）H-和（13）C-核磁共振（NMR）对肝素样品进行评估，并用二维 NMR 对所有峰进行了完全归属。 3-O-磺酸基葡萄糖醛酸残基和 N-磺酸基及 N-乙酰基的百分比范围分别为 5.8-7.9%、78-82%和 13-14%。通过肝素酶 I-III 消化肝素的高效液相色谱（HPLC）-质谱分析，确定了二糖组成存在很大差异。使用肝素酶 I、II 和 III 分别处理后，进行肝素寡糖图谱分析。这些图谱有助于定性和定量识别这些肝素之间的结构差异。通过表面等离子体共振竞争结合测定法，评估了这些肝素与抗凝血酶 III 和凝血酶的结合亲和力。本研究为通用生物工程肝素的合理设计和合成提供了必要的理化性质和活性特征。
