Evaluation of an In Vitro Cell Culture Assay for the Potency Assessment of Recombinant Human Erythropoietin

Abstract

Recombinant human erythropoietin is a sialoglycoprotein that stimulates erythropoiesis. To assess potency of human erythropoietin produced by recombinant technology, we investigated an in vitro TF-1 cell proliferation assay, which was applied in conjunction with a reversed-phase liquid chromatography method for the determination of the content of sialic acids. The results obtained, which were higher than 126.8ng/μg, were compared with those obtained with the in vivo normocythaemic mouse bioassay. The in vitro assay resulted in a non-significant lower mean difference of the estimated potencies (0.61% ± 0.026, p > 0.05). The use of this combination of methods represents an advance toward the establishment of alternative in vitro approaches, in the context of the Three Rs, for the potency assessment of biotechnology-derived medicines.

3r s applied to in vivo biological activity of recombinant human erythropoietin assay

“…To test the biological activity of recombinant erythropoietin, few in vitro methods have been published with the aim of simplifying the assay and overcome ethical issues associated with animal experimentation. The evaluation of proliferative stimulation of an erythroid cell line called UT-7 cell bioassay or reporter UT-7 cell bioassay 6,22 , or the development of an in vitro TF-1 cell proliferation assay for the determination of the content of sialic acids 5 were reported with variable success. Nevertheless, despite all the efforts to apply alternative methods in vitro, the gold standard assay for Ph.…”

“…The approved reference method to define rhEPO potency in laboratories around the world is based on the measurement of the in vivo stimulation of reticulocyte production in normo or polycythaemic mice after the administration of different doses of rhEPO 4 . Despite several efforts being made to develop an alternative method to the in vivo assay 5,6 , the use of laboratory animals is still the gold standard. Meanwhile, minimizing the number of animals used per experiment and refining the appropriate techniques is mandatory, always following an appropriately designed assay to ensure robustness and reproducibility of the method.…”