Development and validation of a UT-7-based in vitro bioassay for potency evaluation of recombinant human erythropoietin

The present study describes the development and validation of a cell-based in vitro bioassay for the potency determination of recombinant human erythropoietin (rHuEPO), proposed as a complementary quality control tool to support post-approval surveillance activities. The bioassay is based on the proliferative response of erythropoietin-sensitive UT-7 erythroleukaemic cells, quantified through a fluorescence-based readout. Method validation was performed in accordance with ICH Q2 guidelines, including the assessment of precision, accuracy, linearity, range, specificity, and robustness, supported by appropriate statistical analysis. The suitability of the assay was evaluated on different commercially available rHuEPO products to assess its analytical performance and fit-for-purpose applicability. According to the European Pharmacopoeia, potency testing of erythropoietins currently relies on in vivo assays performed in normocythaemic or polycythaemic mice, which is the regulatory gold standard due to their ability to capture the overall in vivo biological activity. Nevertheless, this in vitro bioassay represents a practical, validated complementary approach suitable for defined post marketing applications, including batch-to-batch consistency monitoring and official medicine control laboratory activities, supporting the principles of Replacement, Reduction and Refinement (3Rs) in this regulatory testing field. Prospectively, a customized combination of the present animal-free method with orthogonal high-resolution techniques may provide complementary information on the functionality of rHuEPO products, thereby supporting its progressive implementation within an integrated analytical framework.