A Multi-Methodological Approach to Evaluating the Biological Activity of Erythropoietin Marketed in Thailand

บทคัดย่อ

This study evaluated and validated two analytical methods, an enzyme-linked immunosorbent assay (ELISA) and a cell-based bioassay, for the determination of erythropoietin in pharmaceutical formulations marketed in Thailand. Method validation was carried out following ICH guidelines. The ELISA method demonstrated high specificity, as the erythropoietin standard and formulated samples produced clear absorbance signals that were clearly different from those of the excipient matrix. The excipients alone showed minimal response, confirming that formulation components did not interfere with the assay. A strong relationship between absorbance and erythropoietin concentration was observed, and the method showed excellent linearity over the tested range, with coefficients of determination greater than 0.99 across three consecutive days. Accuracy results were acceptable for a biological assay, with recovery values within the expected range. Although some variability was observed in repeatability testing, this was consistent with the nature of immunoassays. Intermediate precision results were consistent across different days and analysts, indicating that the ELISA method is reliable for routine analysis. The cell-based bioassay also showed good specificity. Parallel dilution curves of the erythropoietin standard and sample demonstrated consistent relationships between response and concentration, while formulation excipients produced only weak signals. The assay was linear over a concentration range of 0.0625 to 1 IU/mL, with strong correlations observed over three days. Accuracy testing showed recovery values within acceptable limits, and precision results were within the expected range for cell-based assays, where higher variability is commonly observed. The precision of the assay across different days and analysts confirmed its robustness. In conclusion, both the ELISA and cell-based bioassay methods were successfully validated and shown to be suitable for erythropoietin determination. The ELISA method provides reliable quantitative measurement, while the cell-based assay effectively assesses biological activity and potency. Together, these complementary methods offer a practical and reliable approach for routine quality control of erythropoietin products.