均相时间分辨荧光法测定人胰岛素生物学活性  

Determination of biological activity of human insulin by a homogeneous time-resolved fluorescence method

作　　者：王绿音 杨艳枫 张孝明 吕萍[1] 张慧[1] 李晶[1] 梁成罡[1] WANG Lü-yin;YANG Yan-feng;ZHANG Xiao-ming;Lü Ping;ZHANG Hui;LI Jing;LIANG Cheng-gang(NHC Key Laboratory of Research on Quality and Standardization of Biotech Products,Division of Hormone,National Institutes for Food and Drug Control,Beijing 102629,China;China Pharmaceutical University,Nanjing 211198,China)

机构地区：[1]中国食品药品检定研究院激素室,国家卫生健康委员会生物技术产品检定方法及其标准化重点实验室,北京102629 [2]中国药科大学,江苏南京211198

出　　处：《药学学报》2022年第10期3223-3228,共6页Acta Pharmaceutica Sinica

基　　金：国家科技重大专项课题资助项目(2018ZX09101001-003-004)。

摘　　要：建立基于均相时间分辨荧光免疫分析技术的人胰岛素体外生物学活性测定方法。利用CHO-INSR B1284转基因细胞,通过均相时间分辨荧光免疫分析系统检测人胰岛素的体外生物学活性;对靶细胞铺板密度、药物的量效范围、刺激时间等关键参数进行优化;验证方法的专属性、相对准确度、中间精密度、线性与范围,以及细胞的传代稳定性;以重组人胰岛素国家标准品为参比品,将本研究建立的方法应用于其他人胰岛素类似物原料药、制剂的生物学活性测定。结果表明人胰岛素及其类似物存在剂量效应曲线,R^(2)>0.995,符合四参数方程y=(A-D)/[1+(x/C)^(B)]+D。本方法专属性良好,64%、80%、100%、125%、156%共5个效价浓度的待测品相对效价(n=8)的几何均值、相对偏倚、几何变异系数(GCV,%)均符合《中华人民共和国药典》2020版四部通则9401的要求;且方法可适用于其他胰岛素类似物的生物学活性测定。该方法操作简便、耗时短、准确度高、精密度好,可用于胰岛素类产品的生物学活性评价和质量控制。We developed an in-vitro bioassay for determining the bioactivity of human insulin by homogeneous time-resolved fluorescence immunoassay.CHO-INSR B1284 transgenic cells were used as target cells.Key assay parameters,including the cell density,the range of working concentrations,and the stimulation time were optimized.The specificity,relative accuracy,intermediate precision,linearity,and range of the method were validated,as well as the passage stability of the CHO-INSR B1284 cell line.The national standard of recombinant human insulin was used as the benchmark to evaluate the relative potency of insulin analogues and drugs.The drugs and the reference human insulin showed a dose-response relationship,R^(2)>0.995,which conforms to the four-parameter equation:y=(A-D)/[1+(x/C)^(B)]+D.Specificity of the method was good.The geometric mean,relative bias,and geometric coefficient of variation(GCV,%)of the five concentrations(n=8,64%,80%,100%,125% and 156%)met the requirements of the General Rules of Chinese Pharmacopoeia,2020 edition,Volume IV(9401).In summary,a bioassay for determining the in vitro bioactivity of human insulin based on a homogeneous time-resolved fluorescence technique was established;the method was simple,time-saving,accurate and precise,and could be used for the evaluation of biological activity and quality.

关 键 词：生物学活性 人胰岛素 转基因细胞 均相时间分辨荧光技术 方法学验证 

分 类 号：R917[医药卫生—药物分析学]