Harvard-MIT Division of Health Sciences and Technology, the Koch Institute for Integrative, Cancer Research and the Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Anal Chem. 2011 Oct 15;83(20):7815-22. doi: 10.1021/ac201498a. Epub 2011 Sep 19.
Heparin and the low molecular weight heparins are extensively used as medicinal products to prevent and treat the formation of venous and arterial thrombi. In early 2008, administration of some heparin lots was associated with the advent of severe adverse effects, indicative of an anaphylactoid-like response. Application of orthogonal analytical tools enabled detection and identification of the contaminant as oversulfated chondroitin sulfate (OSCS) was reported in our earlier report. Herein, we investigate whether enzymatic depolymerization using the bacterially derived heparinases, given the structural understanding of their substrate specificity, can be used to identify the presence of OSCS in heparin. We also extend this analysis to examine the effect of other persulfonated glycosaminoglycans (GAGs) on the action of the heparinases. We find that all persulfonated GAGs examined were effective inhibitors of heparinase I, with IC(50) values ranging from approximately 0.5-2 μg/mL. Finally, using this biochemical understanding, we develop a rapid, simple assay to assess the purity of heparin using heparinase digestion followed by size-exclusion HPLC analysis to identify and quantify digestion products. In the context of the assay, we demonstrate that less than 0.1% (w/w) of OSCS (and other persulfonated polysaccharides) can routinely be detected in heparin.
肝素和低分子量肝素被广泛用作药物，以预防和治疗静脉和动脉血栓的形成。2008 年初，一些肝素批次的使用与严重不良反应的出现有关，表明存在类似过敏的反应。正交分析工具的应用使得能够检测和鉴定污染物，正如我们之前的报告中所报道的，这种污染物是过度硫酸化的软骨素硫酸盐（OSCS）。在此，我们研究了是否可以使用细菌来源的肝素酶进行酶解聚合，根据对其底物特异性的结构理解，来识别肝素中是否存在 OSCS。我们还将此分析扩展到检查其他过硫酸盐化糖胺聚糖（GAG）对肝素酶作用的影响。我们发现，所有检查过的过硫酸盐化 GAG 都是肝素酶 I 的有效抑制剂，IC50 值约为 0.5-2μg/mL。最后，利用这种生化知识，我们开发了一种快速、简单的测定方法，使用肝素酶消化后进行大小排阻 HPLC 分析来评估肝素的纯度，以鉴定和定量消化产物。在该测定法的背景下，我们证明在肝素中常规可以检测到低于 0.1%（w/w）的 OSCS（和其他过硫酸盐化多糖）。
