Development and validation of a quantitative cell-based bioassay for comparing the pharmacokinetic profiles of two recombinant erythropoietic proteins in serum.

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.