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PCSK9单抗生物学活性测定方法的建立
PCSK9单抗生物学活性测定方法的建立
刘春雨,朱 磊,张 峰,于传飞,王文波,李 萌,陈 伟,王 兰,高 凯

中国食品药品检定研究院卫生部生物技术产品检定方法及其标准化重点实验室,北京 100050
A method for determination of biological activity of PCSK9 monoclonal antibody
(Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products,Chinese Nationol Institute for Food and Drug Control, Beijing 100050, China)

摘要参考文献相关文章

起始页:1715

摘要:目的:建立前蛋白转化酶枯草溶菌素9(PCSK9)单抗的生物学活性检测方法。方法:利用系列稀释的PCSK9单抗以剂量依赖性方式阻断PCSK9与低密度脂蛋白受体(LDLR)结合,增加低密度脂蛋白(LDL)在HepG2细胞内摄取率,通过BODIPY类荧光染料标记低密度脂蛋白(BODIPY-LDL)检测胞内荧光强度来反映PCSK9单抗的生物学活性,并用PCSK9单抗参比品计算供试品相对百分效价。结果:PCSK9、PCSK9单抗参比品及供试品在该方法中均存在量效关系,且符合四参数方程式:Y=(A-D)/[1+(X/C)B]+D。利用该法对4批PCSK9单抗供试品进行检测,每批供试品重复试验3次,PCSK9单抗的相对百分效价的平均值在94.25%~110.77%之间,变异系数均小于15%。结论:该方法灵敏度高、重复性好、结果准确,可作为PCSK9单抗生物学活性测定的常规检测方法。

关键词:单克隆抗体;前蛋白转化酶枯草溶菌素9(PCSK9);低密度脂蛋白(LDL);低密度脂蛋白受体(LDLR);生物学活性;荧光染色

通讯作者:王兰,女,博士,副研究员,主要从事抗体类生物治疗药物质量控制研究。

基金项目:国家“重大新药创制”科技重大专项(2014ZX09304311-001;2012ZX09304010)

作者简介:刘春雨,女,硕士,助理研究员,主要从事抗体类生物治疗药物质量控制研究。

Abstract:Objective: To establish a method for determination of biological activity of proprotein convertase subtilisin-like kexin type 9 (PCSK9) monoclonal antibody (mAb). Methods: Serially diluted PCSK9 mAb was used to block the binding of PCSK9 to low-density lipoprotein receptor (LDLR) in a dose-dependent manner, thus mediating the uptake rate of low-density lipoprotein (LDL). Then LDL was labeled with BODIPY, and the biological activity of PCSK9 mAb was determined by measuring the intracellular fluorescence intensity. The percent relative potency of PCSK9 mAb was calculated by comparison with the reference substance. Results: PCSK9, PCSK9 mAb and the reference substance showed dose-response effect which complied with the 4-parameter equation: Y=(A-D)/[1+(X/C)B]+D. Four batches of final products were tested for three times. The mean percent relative potency was 94.25%~110.77%, and the coefficient of variation was less than 15%. Conclusion: The method was sensitive, accurate and reliable, which can be used as the routine method for determination of biological activity of PCSK9 mAbs.

Key words:monoclonal antibody; proprotein convertase subtilisin-like kexin type 9(PCSK9); low-density lipoprotein (LDL); low-density lipoprotein receptor (LDLR); biological activity; fluorescence dye



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