降纤酶关键质量属性研究  

Study on key quality properties of defibrase

作　　者：邹剑[1,2] 李炯 杨金亮[1] 李炎[2] 杨蕾 马晶 袁月 ZOU Jian;LI Jiong;YANG Jin-liang;LI Yan;YANG Lei;MA Jing;YUAN Yue(State Key Laboratory of Biotherapy,Sichuan University,Chengdu 610044,China;Sichuan Institute for Drug Control,NMPA Key Laboratory for Quality Control and Evaluation of Vaccines and Biological Products,SCMPA Key Laboratory for Quality Control Monitoringund Risk Assessment of Biological Products,Chengdu 611731,China)

机构地区：[1]四川大学生物治疗国家重点实验室,成都610044 [2]四川省药品检验研究院、国家药品监督管理局疫苗及生物制品质量控制与评价重点实验室,四川省药品监督管理局生物制品质量监测与风险评估重要实验室,成都611731

出　　处：《药物分析杂志》2023年第12期2137-2146,共10页Chinese Journal of Pharmaceutical Analysis

基　　金：国家药典委员会标准提高课题(2018H060)。

摘　　要：目的:通过对降纤酶鉴别、纯度、分子量、等电点、N端序列、比活性6个方面关键质量属性的表征,为后续标准提高提供依据。方法:采用SDS-PAGE电泳法分析不同来源的蛇毒原料;采用紫外-可见分光光度法、SDS-PAGE电泳法、体积排阻色谱和反相超高效液相色谱法、基质辅助激光解析电离-飞行时间质谱法、等电聚焦电泳法、“edman”N端测序法以及凝固法和生色底物法对不同企业提供的降纤酶进行测定和分析。结果:不同来源的尖吻蝮蛇蛇毒蛋白组成基本一致,尖吻蝮蛇蛇毒和白眉蝮蛇蛇毒蛋白质组成差异较大。降纤酶对苯甲酰-L-精氨酸甲酯盐酸盐最大吸收波长为247 nm,对苯甲酰-L-精氨酸乙酯盐酸盐最大吸收波长为254 nm,但对苯甲酰-DL-精氨酰对硝基苯胺无水解作用。降纤酶能完全水解纤维蛋白原α肽链,活性受到苯甲基磺酰氟、二硫苏糖醇和对氨基苯甲醚的抑制。尺寸排阻高效液相色谱法降纤酶纯度测定结果高于反向超高效液相色谱法,前者为99.9%,后者为95.4%。SDS-PAGE电泳法测降纤酶完整分子量受标准品线性范围的影响,采用10~250 kDa范围的标准品点拟合,降纤酶完整分子量为(42.1±1.6)kDa;采用10~100 kDa范围的标准品点拟合,降纤酶分子量为(38.4±1.3)kDa。MALDI-TOF-MS法测得降纤酶完整分子量为(33.3±0.5)kDa,脱糖基分子量为(28.8±0.02)kDa。降纤酶pI值在3.0~4.0之间,N端序列为VIGGVECDINEHRFL。凝固法和生色底物法测比活性相关性显著(P<0.01),但凝固法结果高于生色底物法。结论:不同企业提供的降纤酶的6个方面的关键质量属性基本一致。现行降纤酶药品国家标准中鉴别、纯度、分子量等项目有待进一步增修订。Objective:To provide the basis for the improvement of the standard by characterizing the key quality attributes of defibrase in six aspects:identification,purity,molecular weight,isoelectric point,N-terminal sequence and specific activity.Methods:The raw materials of snake venom from different sources were analyzed by SDS-PAGE electrophoresis.Ultraviolet-visible spectrophotometry,SDS-PAGE electrophoresis,size exclusion chromatography and reverse phase ultra-high performance liquid chromatography,matrix assisted laser desorption ionization-time of flight mass spectrometry,isoelectrofocusing electrophoresis,“edman”N-terminal sequencing method,coagulation method and color substrate method were used to determine and analyze the defibrase supplied by different manufacturers.Results:The venom composition of Deinagkistrodon acutus from different sources was basically the same,and the venom composition of Deinagkistrodon acutus was different from that of Gloydius ussuriensis.The maximum absorption wavelength of defibrase was 247 nm for benzoyl-L-arginine methyl ester hydrochloride and 254 nm for benzoyl-L-arginine ethyl ester hydrochloride,but no hydrolysis was observed for benzoyl-DL-arginine p-nitroaniline.Defibrase could completely hydrolyze fibrinogenαpeptide chain,and the activity is inhibited by phenylmethylsulfonyl fluoride,dithiothreitol and p-aminoanisole.The purity of defibrase by size exclusion HPLC and RP-UPLC were 99.9%and 95.4%,respectively.The complete molecular weight of defibrase measured by SDS-PAGE electrophoresis was affected by the linear range of standard substance.The complete molecular weight of defibrase was(42.1±1.6)kDa by standard point fitting in the range of 10-250 kDa,as well as(38.4±1.3)kDa by standard point fitting in the range of 10-100 kDa.The molecular weight of defibrase was(33.3±0.5)kDa and that of deglycosylation was(28.8±0.02)kDa by MALDI-TOF-MS method.The pI values of defibrase were between 3.0 and 4.0,and the N-terminal amino acid sequence was VIGGVECDINEHRFL.The specific

关 键 词：尖吻蝮蛇 白眉蝮蛇 蛇毒 降纤酶 SDS-PAGE电泳 尺寸排阻色谱 反向色谱 纤维蛋白原 分子量 等电点 N端序列 比活性 

分 类 号：R917[医药卫生—药物分析学]