用耗尽型肝素酶消化法和强阴离子交换色谱法对肝素进行定量成分分析

Pierre Mourier, Pascal Anger, Céline Martinez, Fréderic Herman, Christian Viskov
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引用次数: 33

摘要

肝素是一种线性硫酸酸化多糖,作为抗凝剂广泛应用于治疗。它也是制造低分子肝素(LMWH)的原料。肝素和低分子肝素的质量控制对于确保最终产品的安全性和治疗活性至关重要。然而,由于其复杂和异质结构,需要正交分析技术来表征肝素的组成部分。最先进的肝素分析方法之一是基于使用肝素酶I, II和III的混合物的完全酶消化,然后通过液相色谱分离得到的低聚糖。欧洲药典强阴离子交换色谱法用于定量依诺肝素中的1,6-无羟基衍生物,这里应用于肝素构建块的分析。它们的定量,即测定它们在肝素链中的平均w/w百分比,是在鉴定了包括糖丝氨酸衍生物和3-O硫酸二糖和四糖在内的所有成分后得到的。因此,这项工作提供了对未分离肝素的构建块的全面概述,包括那些通过制造过程进行化学修饰的肝素,无论是在多糖链内还是在其还原端。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Quantitative compositional analysis of heparin using exhaustive heparinase digestion and strong anion exchange chromatography

Heparin is a linear sulfated polysaccharide widely used therapeutically as an anticoagulant. It is also the starting material for manufacturing low-molecular-weight heparins (LMWH). Quality control of heparin and LMWH is critical to ensure the safety and therapeutic activity of the final product. However due to their complex and heterogeneous structure, orthogonal analytical techniques are needed to characterize the building blocks of heparin. One of the state-of-the-art methods for heparin analysis is based on complete enzymatic digestion using a mixture of heparinases I, II, and III, followed by the separation of the resulting oligosaccharides by liquid chromatography. The European Pharmacopoeia strong anion-exchange chromatographic method, used to quantify 1,6-anhydro derivatives in enoxaparin, is here applied to the analysis of the heparin building blocks. Their quantification, namely the determination of their average w/w percentage in the heparin chain, is obtained after identification of all components including glycoserine derivatives and 3-O sulfated di- and tetrasaccharides. This work therefore provides a comprehensive overview of the building blocks of unfractionated heparin, including those chemically modified by the manufacturing process, either within the polysaccharide chain or at its reducing end.

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