Avaliação comparativa de potência de eritropoietina humana recombinante por bioensaio alternativo e correlação com métodos físico-químicos

Abstract

Erythropoietin is a sialoglycoprotein which promotes the increase of erythropoiesis. Clinically is used for the treatment of anaemia associated to chronic renal failure. Identification and separation of isoforms of recombinant human erythropoietin (rhEPO) in biopharmaceuticals of different origins, was carried out by isoelectric focusing (IEF) western blotting and, also by lectin binding with Triticum vulgaris, showing 4-7 isoforms distributed in the isoeletric range of 4.4 to 5.2. N-acetylneuraminic acid content was quantified by reversed-phase liquid chromatography method with fluorescence detection giving values higher than 108.74 ηg/μg. Biological activity was evaluated by the normocythaemic mice bioassay, and investigating the TF-1 cell line in vitro. The correlation of the results of both of the methods were significant, as calculated by the Pearson s coefficient (r = 0.9967). In addition, the content/potency of the biopharmaceutical products was assessed by validated reversed phase and size exclusion liquid chromatography methods, showing mean values 2.11% and 1.21% lower, respectively, related to the in vivo bioassay. Sample was degraded under UV light to generate deamidate/sulphoxide forms and treatment at 65ºC for 12 hours to produce dimeric and aggregated forms. The potencies were evaluated by the normocythaemic mice assay and the TF-1 cell culture assay giving mean reduction of 14.05% and 32.87%, respectively, related to the intact molecule. The alternative in vitro assay investigated in the context of the reduction or replacement of the animals, and the evaluation of the correlations between physicochemical and biological methods, represent improvements which can be applied to the production steps and for the quality control of rhEPO, contributing to ensure the batch-to-batch consistency of bulk and finished biological products.