不同来源肝素酶Ⅰ对依诺肝素钠降解作用的研究

Degradation characteristics of enoxaparin sodium by heparinaseⅠfrom various sources

目的:探索不同来源的肝素酶Ⅰ对依诺肝素钠降解特性的差异,以及依诺肝素钠精细结构表征,为此类药物的质量控制提供理论依据。方法:考察3种肝素酶Ⅰ在不同条件下对依诺肝素钠的降解特点,并利用离子交换色谱法纯化降解产物,之后进行质谱和核磁共振分析,获得结构信息,从而阐述降解产物组成和降解效率的差异。结果:不同来源的肝素酶Ⅰ对依诺肝素钠的降解效果不同。大肠杆菌表达体系的融合蛋白肝素酶Ⅰ的降解效率要高于来源于肝素黄杆菌的原生肝素酶Ⅰ,而大肠杆菌表达体系的重组肝素酶Ⅰ效率最低。经离子交换纯化获得数个寡糖,通过结构确认的1个寡糖组分(ΔUA2S-Glc NS6S-Ido A2SGlcNS6S)在第1和第2种酶的降解产物中没有发现,而在第3种酶的降解产物中始终存在。结论:本文比较了3种不同来源肝素酶Ⅰ降解依诺肝素钠过程中的细微差异,为以肝素酶降解为基础的依诺肝素钠结构研究和质量控制提供了有效支持。

Objective:To explore the difference in the degradation characteristics of enoxaparin sodium by heparinaseⅠfrom various sources,and the fine structure characterization of enoxaparin sodium,to provide a theoretical basis for the quality control of such drugs.Methods:The degradation characteristics of three heparinaseⅠto enoxaparin sodium under different conditions were investigated,and the degradation products of enoxaparin were subsequently purified by ion exchange chromatography,and mass spectrometry and NMR analysis were carried out to obtain structural information,so as to expound the differences in degradation product composition and degradation efficiency.Results:Different sources of heparinaseⅠshowed different efficiency on enoxaparin sodium.The degradation efficiency of the fusion protein heparinaseⅠof the E.coli expression system is higher than that of the native heparinaseⅠderived from Flavobacterium heparinum,while the recombinant heparinaseⅠfrom E.coli expression system showed with the lowest efficiency.Several oligosaccharides were obtained by ion exchange purification,and one oligosaccharide component(ΔUA2 S-GlcNS6 S-IdoA2 S-GlcNS6 S),which was confirmed by structure,was absent in the degradation products of the first and second enzymes,but was present in which of the third all the times.Conclusion:This article compares the subtle differences of the degradation of enoxaparin sodium by heparinase I from three difference sources,which shall provide effective support for the structural research and quality control of enoxaparin sodium based on heparinase degradation.