Avaliação da atividade e caracterização de eritropoietina humana recombinante em produtos farmacêuticos

Abstract

RESUMORealizou-se a identificação de eritropoietina humana recombinante em produtos farmacêuticos comerciais por eletroforese em gel de poliacrilamida e detecção com anticorpos específicos, demonstrando-se típi-ca banda larga, semelhante ao padrão de rhEPO da Farmacopéia Européia. Igualmente por focalização isoelétrica e imunodetecção, observaram-se 5-6 isoformas características de acordo com o laboratório produtor. A avaliação de potência efetuada através de ensaio biológico em camundongos normocitêmicos forneceu valores entre 67,6% e 119,4% em relação à declarada. A precisão dos ensaios combinados, calculada pela ponderação, variou de 200 a 389. Os testes de endotoxinas bacterianas, toxicidade e pH apresentaram valores variáveis conforme o lote. Concluiu-se destacando a importância dos testes e ensaios de controle para assegurar a qualidade lote a lote e garantir a eficácia terapêutica dos produtos farmacêuticos. The identification of rhEPO in pharmaceutical products was carried out by polyacrylamide gel electrophoresis and detection with specific antibodies that revealed a typical single broad band similar to that obtained with the European Pharmacopoeia Biological Reference Preparation for erythropoietin. The isoelectric focusing with immunodetection revealed extensive heterogeneity with 5-6 isoforms, characteristic according to the manufacturer. The potency was assessed by the normocythaemic mouse assay with values within 67.6% and 119.4% calculated against the stated potency. The precision evaluated by the weight was within 200 and 389, for the combined assays. The toxicity, bacterial endotoxins, and pH gave variable results according to the batch. In conclusion, we emphasize the importance of the batch-to-batch tests and assays that would guarantee the quality and therapeutic efficacy of the pharmaceutical products.

Potency Evaluation of Recombinant Human Erythropoietin in Brazil: Assessment of Reproducibility Using a Practical Approach

“…Although it is possible to combine valid results obtained in independent potency determination tests performed upon batch release of rhEPO (Council of Europe 2012;USP 2012a;WHO 2013), in this study, all the results of INCQS were obtained from single assays because there was no need for combination trials of the satisfactory results in the samples tested. These data differ from those of previous studies, where it was necessary to combine two or even three independent experiments to obtain satisfactory results for the same sample in different strains, such as in Swiss Webster, B6D2F1, CF1, or BAlb/c mice (Albertengo et al 1999;Schmidt et al 2003;Lopes 2004;Barth et al 2008;Costa et al 2010;Silva et al 2013). This is probably due to the optimization achieved at our institute in terms of standardization and control factors (such as the range of body weight and age of the animals, ambient humidity and temperature of the animal facilities, precision in sample preparation and reading of reticulocytes, and use of calibrated instruments) that could affect the outcome of a trial, thereby reducing the variability inherent in the assay.…”

“…EPO is produced in adults mainly by the renal cortex, and its main function is regulation of erythropoiesis (Jacobson et al 1957;Fried 1972;Zanjani et al 1981;Fisher et al 1996;Schmidt et al 2003). In 1977, EPO was extracted and purified from the urine of anemic patients.…”

“…Method B is normally the method of choice because method A inflicts greater stress and suffering on the animals that must be kept for long periods in hypobaric chambers and exposed to radioisotopes (ECVAM 2002). Therefore, different strains of normocythaemic mice have been tested to evaluate rhEPO potency, such as CF1, Balb/c, Swiss Webster, NIH, C57BL6 and B6D2F1 strains (Albertengo et al 1999;Schmidt et al 2003;Barth et al 2008;Council of Europe 2011;Silva et al 2013). The process of batch release of certain biological products, such as rhEPO, is generally characterized by the use of a large number of animals.…”

Biosimilar epoetins and other “follow‐on” biologics: Update on the European experiences

“…While clinical trials showed equivalence of copied rhEPOs from Cuba [104], China [105] or Korea [106,107], the identity and purity of some of the medicines was found to be less sufficient [108,109]. The purported copies of rhEPOs from Korea, India and China contained more glycoforms and other impurities than the originator's epoetin alfa (Epogen 1 , Amgen).…”

“…Most importantly, covalent aggregates were detected in some of the copied products [108]. Another study identified copied epoetins that were contaminated with endotoxin [109]. There are two reports on the in vivo activity of such products.…”