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首页> 外文期刊>Journal of Pharmaceutical and Biomedical Analysis: An International Journal on All Drug-Related Topics in Pharmaceutical, Biomedical and Clinical Analysis >Development and validation of a quantitative cell-based bioassay for comparing the pharmacokinetic profiles of two recombinant erythropoietic proteins in serum.
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Development and validation of a quantitative cell-based bioassay for comparing the pharmacokinetic profiles of two recombinant erythropoietic proteins in serum.

机译：一种基于细胞的定量生物测定方法的开发和验证，用于比较血清中两种重组促红细胞生成素蛋白的药代动力学特征。

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摘要

An in vitro cell-based bioassay was developed and validated to assess the pharmacokinetic profiles of two novel therapeutic recombinant proteins (EP1 and EP2) with erythropoiesis stimulating properties in Sprague-Dawley rats. While immunoassays are the standard choice for evaluating the pharmacokinetic parameters of drugs, no immunoassay was available for EP2, necessitating the need for a quantitative bioassay capable of measuring both EP1 and EP2 separately so that appropriate comparisons could be made. The bioassay described here utilizes a sub clone of the murine 32D cell line transfected with the gene encoding for the human erythopoietin (HuEPO) receptor. Erythropoietin (EPO), EP1 and EP2 exert their proliferative effect on the cell line by signaling through the HuEPO receptor. The proliferation induced by the erythropoietic proteins was measured by [methyl-(3)H]thymidine incorporation into the cellular DNA. The assay was conducted in 96-well microtiter plates and had relatively high throughput. The Guidelines of the International Conference on Harmonization (ICH) were followed for the validation of the different assay parameters including robustness, linearity, accuracy, precision, limit of quantitation (LOQ) and specificity. The robustness of the bioassay is demonstrated by the lack of an effect of age of the 32D cell culture on the performance of the EP2 bioassay. The bioassay demonstrated good linearity, yielding a coefficient of determination of 0.99 or higher for both EP1 and EP2. The assay showed reproducible dose-response curves for EP1 in the range of 0.039-2.5 ng/mL and for EP2 in the range of 0.125-8 ng/mL. The accuracy estimates ranged between 98% and 108% for EP1 and between 90% and 110% for EP2 in the reproducible range mentioned above. Intermediate precision (within-plate R.S.D.) in the same range was within 26% and 17% for the EP1 and EP2 bioassays, respectively. The validated bioassays for EP1 and EP2 were utilized to quantitatively analyze serum samples from a pharmacokinetic study conducted to compare the profiles of the two compounds in Sprague-Dawley rats.
机译：开发了一种基于体外细胞的生物测定法，并进行了验证，以评估两种新的具有重组红细胞生成特性的治疗性重组蛋白（EP1和EP2）在Sprague-Dawley大鼠中的药代动力学特征。虽然免疫测定是评估药物药代动力学参数的标准选择，但没有针对EP2的免疫测定，因此需要能够同时测量EP1和EP2的定量生物测定，以便可以进行适当的比较。本文所述的生物测定法利用转染了编码人类促红细胞生成素（HuEPO）受体的基因的鼠32D细胞系的亚克隆。促红细胞生成素（EPO），EP1和EP2通过HuEPO受体发出信号，从而在细胞系中发挥增殖作用。通过[甲基-（3）H]胸苷掺入细胞DNA中来测量由促红细胞生成蛋白诱导的增殖。该测定法在96孔微量滴定板中进行，通量较高。遵循国际协调会议（ICH）的指南来验证不同的测定参数，包括鲁棒性，线性，准确性，精密度，定量限（LOQ）和特异性。通过缺乏32D细胞培养物的年龄对EP2生物测定性能的影响来证明生物测定的稳健性。该生物测定法显示出良好的线性，对EP1和EP2的测定系数为0.99或更高。该测定显示出EP1的可重现剂量反应曲线在0.039-2.5 ng / mL范围内，EP2的可重现的剂量反应曲线在0.125-8 ng / mL范围内。在上述可重复范围内，EP1的准确度估计值介于98％和108％之间，EP2的准确度估计值介于90％和110％之间。 EP1和EP2生物测定法在相同范围内的中间精度（板内R.S.D.）分别在26％和17％之内。经验证的EP1和EP2生物测定法可用于定量分析药代动力学研究中的血清样品，以比较两种化合物在Sprague-Dawley大鼠中的分布。

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