降纤酶质量研究

Quality Research on Defibrase

目的:考察降纤酶的质量现状及存在问题。方法:对国家食品药品监督管理总局(CFDA)网站数据库上检索到的有降纤酶生产批文的生产企业进行问卷调查,对部分生产企业进行现场调研,针对上述两个环节中发现的问题,对降纤酶原液、中间体、半成品、成品开展了降纤酶原料(液)蛋白质序列分析等一系列实验室探索性研究,提升其安全性、有效性、稳定性和可控性。结果:目前市场上流通的大部分样品的纯度均较低,其中尖吻蝮蛇降纤酶的主要成分为蕲蛇酶,白眉蝮蛇降纤酶的主要成分为NCBI蛋白数据库中编号为gi|143681919|(sp|P85109.1|)的蛋白;降纤酶效价测定影响因素中Tris的浓度影响最大;加入大分子保护剂可以改善样品中的辅料右旋糖酐对效价测定结果的影响。效价测定中两种仪器法(磁珠法和光学法)测定的结果差距较大,均比目测法低,目前尚不适用于降纤酶效价的测定。热稳定性表明注射用降纤酶热稳定性较好,降纤酶注射液的热稳定性较差。本研究采用浓缩一倍的Tris缓冲液稀释纤原,并加入适当的保护剂,建立了降纤酶半成品效价测定的方法。结论:目前降纤酶的质量有待提高。两种蛇毒来源的降纤酶结构、纯度等差别较大,现行的标准项目设置不全面;缺少专属性较强目前的鉴别;异常毒性、效价测定可操作性差等,不能有效地控制降纤酶的质量。有必要将尖吻蝮蛇和白眉蝮蛇来源的降纤酶作为两个品种管理,同时加强降纤酶的标准化研究。

Objective： To research the quality level and problems of domestic defibrase. Methods： A questionnaire survey was performed in the factories with the production approval on the website database of CFDA, and a site survey was carried out in some of the factories. For the problems found out in the two surveys, a series of exploratory lab studies on the basis of ordinary analysis such as sequencing of proteins were performed on the materials, intermediates, semi-finished products and final products in order to improve their safety, validity, stability and controllability. Results：The purity of the most of the marketed products was low. Acutobin was the principal component in defibrase from Agkistrodon acutus, and the major ingredient in defibrase from Agkistrodon halys ussuriensis was an unknown protein, which was numbered by gi ｜ 143681919 ｜ （sp ｜ P85109. 1 ｜） in NCBI protein database. Tris concentration was the main influencing factor in the test of biological potency. Macromolecular substances could decrease the effect of dextran. A new method for the test of biological potency of defibrase semi-products was developed by diluting the fiber with double enriched Tris buffer and adding the appropriate macromolecular substances. The results respectively measured by optical and magnetic method were notably different and both lower than that of the standard method, therefore, they were not suitable for the biological potency test of defibrase. Defibrase injection had poorer thermo-stability than defibrase for injection. Conclusion： The risk of defibrase is huge because of the poor quality. Defibrase from Agkistrodon acutus is notably different from that from Agkistrodon halys ussuriensis in protein structure and purity. The current standard method without the items including identification and specificity can not comprehensively evaluate the quality of defibrase. The operability is poor in the abnormal toxicity examination and biological potency test. It is necessary to intensify the supervision of defibrase from Agkistrodon acutus and Agkistrodon halys ussuriensis as two kinds of products. Meanwhile, the stand-ardization research on defibrase should be enhanced.