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.
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)
出处
《中国生物制品学杂志》
CAS
CSCD
北大核心
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)。
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
Recombinant human interleukin-1 receptor antagonist(IL-1Ra)
Human melanoma cells
Biological activity
Cell proliferation
