HL-60/NF-κB报告基因热原检测法在单抗药品热原检测中的应用

Application of HL60/NF-κB Pyrogen Reporter Gene Assay in Detection of Pyrogen in Monoclonal Antibodies

初步建立了一种全新的人早幼粒白血病(HL-60)/核转录因子-κB(NF-κB)报告基因热原检测法,探讨该方法在阿达木单抗注射液、贝伐珠单抗注射液、托珠单抗注射液和达雷妥尤单抗注射液热原检测中的应用,并对该方法进行准确度、重复性和灵敏度验证。在4种单抗注射液无干扰最小稀释倍数下配制不同浓度的脂多糖(LPS)标准品溶液作用于细胞,以LPS浓度对数值为横坐标,化学光强度值为纵坐标,拟合标准曲线。结果显示,R^(2)均大于0.9500,当LPS标准品浓度大于1 EU/ml时,变异系数小于20%,方法检测限平均值为0.02 EU/ml,为生物制品药物热原检测新方法的拓展提供了参考。

A new human promyelocytic leukemia(HL-60)/nuclear factor-κB(NF-κB)reporter gene pyrogen detection method was established initially,and its application in pyrogen detection in adalimumab injection,bevacizumab injection,tocilizumab injection and daratumumab injection was discussed.And the accuracy,repeatability and sensitivity of this method were validated.The lipopolysaccharide(LPS)standard solutions with different concentrations were prepared to act on the cells under the minimum dilution factor of the four monoclonal antibody injections without interference.The logarithmic value of LPS concentration was taken as the abscissa and the chemiluminescence intensity value was taken as the ordinate,and a standard curve was fitted.The results showed that the R^(2) values were all greater than 0.9500.When the concentration of LPS standard was greater than 1 EU/ml,the coefficient of variation was less than 20%,and the average detection limit of this method was 0.02 EU/ml,which provided a reference for the expansion of the pyrogen detection method for biological products.