重组人白细胞介素-1受体拮抗剂生物学活性检测方法的建立及验证  

Development and verification of a method for determination of biological activity of recombinant human interleukin-1 receptor antagonist

作　　者：俞露 王莹 张宇 刘莹 刘景会 刘玉林 YU Lu;WANG Ying;ZHANG Yu;LIU Ying;LIU Jing-hui;LIU Yu-lin(Department of Cytokines,Changchun Institute of Biological Products Co.,Ltd.,Changchun 130012,Jilin Province,China)

机构地区：[1]长春生物制品研究所有限责任公司细胞因子室,吉林长春130012

出　　处：《中国生物制品学杂志》2021年第11期1370-1374,共5页Chinese Journal of Biologicals

基　　金：吉林省科技发展计划项目(20160204034YY)。

摘　　要：目的建立重组人白细胞介素-1受体拮抗剂(interleukin-1 receptor antagonist,IL-1Ra)生物学活性检测方法,并对方法进行验证。方法根据IL-1Ra阻断IL-1β杀伤人黑色素瘤细胞A375.S2的能力评价IL-1Ra的生物学活性,结果用标准品进行校正。对方法的样品起始浓度(8、10、12、14μg/mL)和IL-1β终浓度(2、4、6 ng/mL)进行优化,并验证方法的专属性、准确度、线性范围、中间精密度、耐用性。用建立的方法检测本公司在研产品3批原液和3批成品的生物学活性。结果确定该方法的样品起始浓度为8μg/mL,IL-1β终浓度为4 ng/mL。IL-1Ra原液缓冲液成分及成品中辅料成分对检测结果无影响;线性范围为6~13μg/mL,相关系数R2为0.9921;准确度验证线性回归方程的斜率为0.9758;不同试验人员于不同时间对1批原液、1批成品检测结果的几何变异系数(geometric coefficient of variation,GCV)分别为13.895%和8.670%,均<20%;不同细胞代次、不同细胞密度下相对效价测定值的GCV为5.595%,<20%。用建立的方法检测3批原液和3批成品的结果较稳定。结论成功建立了重组人IL-1Ra生物学活性检测方法,该方法准确度高,精密度好,耐用性强,检测结果稳定可靠,可用于产品的生物学活性评价和质量控制。Objective To develop and verify a method for determination of biological activity of recombinant human interleukin-1 receptor antagonist(IL-1Ra).Methods The biological activity of IL-1Ra was evaluated according to its ability in blocking the killing effect of interleukin-1β(IL-1β)on human melanoma A375.S2 cells,and the result was corrected with the standard.The initial sample concentration(8,10,12 and 14μg/mL)and final IL-1βconcentration(2,4 and 6 ng/mL)were optimized,and the method was verified for specificity,accuracy,linear range,intermediate precision and durability.Three batches of bulks and three batches of final products of IL-1Ra prepared by Changchun Institute of Biological Products Co.,Ltd.were determined by the developed method.Results The initial sample concentration and final IL-1βconcentration of the developed method were optimized as 8μg/mL and 4 ng/mL respectively.The components of buffer in bulk and subsidiary materials in final product showed no impact on the determination result.The linear range of the developed method was 6~13μg/mL,with a correlation coefficient(R2)of 0.9921.The slope of linear regression equation in verification for accuracy was 0.9758.The geometric coefficients of variation(GCVs)of a batch of bulk and a batch of final product by different persons at different time points were 13.895%and 8.670%respectively,both of which were less than 20%.However,the GCV of determination result of relative potencies with cells of different passages and densities wsa 5.595%,which was less than 20%.The determination results of three batches of bulks and three batches of final products by the developed method were relatively stable.Conclusion A method for determination of biological activity of recombinant human IL-1Ra was successfully developed,which showed high accuracy,precision and durability,and might be used for the biological activity evaluation and quality control of the product.

关 键 词：重组人白细胞介素-1受体拮抗剂 人黑色素瘤细胞 生物学活性 细胞增殖 

分 类 号：Q331[生物学—遗传学]