目的:利用时间分辨荧光免疫分析系统建立人胰岛素基于转基因细胞的生物学活性检测方法。方法:以CHO-INSRB1284转基因细胞2.5×105个·mL-1作为靶细胞,以400 pmol·mL-1作为初始浓度对人胰岛素进行3倍系列稀释,随后药物与靶细胞作用20 min,通过时间分辨荧光免疫分析系统,进行生物学活性检测,对该方法进行关键参数的优化,并依据2020年版《中华人民共和国药典》四部通则9401和ICH指导原则Q2(R1)、Q6B进行验证。结果:人胰岛素在本方法中存在良好的量效关系,符合四参数方程:y=(A-D)/[1+(x/C)B]+D。本方法专属性强,5个效价水平(64%、80%、100%、125%及156%)的相对生物学活性(n=8)的几何平均值分别为(55.8±2.06)%、(82.1±5.52)%、(94.4±5.46)%、(121.4±5.94)%、(154.4±8.37)%,相对偏倚及其90%置信区间分别为 -12.8%(-16.3%, -9.9%)、2.6%(-4.4%, 9.5%)、-5.6%(-11.2%, -0.3%)、-2.9%(-7.7%, -1.8%)、-1.0%(-7.1%,3.9%),相对偏倚及置信区间的绝对值均不超过20%,表明该方法相对准确度良好。5个效价水平的几何变异系数(GCV)及其置信区间的上限均小于20%,结果证明此方法精密性较好。线性范围为64%～156%,斜率为1.031,R2大于0.98。结论:本研究利用时间分辨荧光免疫分析系统成功建立了人胰岛素基于转基因细胞的生物学活性检测方法,该方法可用于人胰岛素体外药效评价和质量控制。
Objective: To establish a method for detecting the biological activity of human insulin against transgenic cells using a time-resolved fluorescence immunoassay system. Methods: CHO INSRB 1284 transgenic cells with 2.5×105 cells·mL-1 were used as target cells. The initial concentration of 400 pmol·mL-1was used to dilute human insulin in a series of 3 times. Then the drug was treated with the target cells for 20 min, biological activity was detected by time-resolved fluorescence immunoassay system. Key parameters of the method were optimized and verified according to the 2020 edition of the Chinese Pharmacopoeia, the four general rules 9401 and the ICH guidelines Q2(R1)、Q6B for validation. Results: The method shows good dose-response relationship, which conforms to the four-parameter equation: y=(AD)/[1+(x/C)B]+D. The method had a strong specificity. The geometric mean of the relative biological activity of the 5 potency levels(64%, 80%, 100%, 125% and 156%)(n=8) was respectively (55.8±2.06)%, (82.1±5.52)%, (94.4±5.46), (121.4±5.94)% and (154.4±8.37)%, the relative bias and its 90% confidence interval were respectively -12.8% (-16.3%, -9.9%), 2.6% (-4.4%, 9.5%), -5.6% (-11.2%, -0.3%), -2.9% (-7.7%, -1.8%) and -1.0% (-7.1%, 3.9%). The absolute value of relative bias and confidence interval did not exceed 20%, indicating that the method was relatively accurate. The geometric coefficient of variation(GCV) and the upper limit of its confidence interval for the five potency levels were all less than 20%. The results showed that the proposed method has good precision. The linear range was 64%-156%, the slope was 1.031, and the R2 was greater than 0.98. Conclusion: The time-resolved fluorescence immunoassay system is used to successfully establish the biological activity detection method of human insulin based on transgenic cells, which can be used for efficacy evaluation in vitro and quality control.
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