Dissertação
Avaliação de eritropoietina humana recombinante por métodos cromatográficos validados e correlação com o bioensaio
Date
2009-03-13Registration in:
FERRETTO, Ricardo Machado. Assessment of recombinant human erythropoietin of
chromatographic method validated and correlation
of bioassay. 2009. 77 f. Dissertação (Mestrado em Farmacologia) - Universidade Federal de Santa Maria, Santa Maria, 2009.
Author
Ferretto, Ricardo Machado
Institutions
Abstract
Erythropoietin is a endogenous glycoprotein which stimulates the erythropoiesis. Clinically is used for the treatment of renal anaemia. The chromatographic method for the potency evaluation of recombinant human erythropoietin (rhEPO) in pharmaceutical products was validated in the present work. A size-exclusion liquid chromatography method (SE-LC) was validated using a BioSep-SEC-S
2000 column (300 mm x 7,8 mm I.D.), maintained at ambient temperature (25°C). The mobile phase consisted of 0.001 M monobasic potassium phosphate, 0.008 M dibasic sodium phosphate and 0.2 M sodium chloride buffer, pH 7.4, run isocratically at a flow rate of 0.5 mL/min with detection at 214 nm. The chromatographic separation was obtained within 30 min and it was linear in the concentration
range of 5-150 μg/mL (r2=0.9991). Validation parameters were evaluated such as sensitivity, precision, accuracy, detection limit, quantitation limit and robustness. The specificity was evaluated by the peak purity of the Recombinant Human Erythropoietin Biological Reference Preparation of European Pharmacopoeia (rhEPO-SBR) storage under stress conditions. The proposed method was
applied for the analysis of erythropoietin in pharmaceutical products, evaluating also the dimmers and
high-molecular-mass forms. Moreover, was performed the analysis of erythropoietin by reversedphase liquid chromatography (RP-LC), evaluating the sulphoxides and deamidates forms. The correlation with the methods was established, showing mean differences between the estimated
potencies of 2.50% lower for the bioassay, and 9.29% higher for the RP-LC, compared with the sizeexclusion
liquid chromatography method, with significative correlation, conform calculated for the Pearson s correlation coefficient (r=0,9629 e r=0,9422, respectively). The alternative established represents a contribution towards the reduction or replacement of
the animals improving the quality control and assuring the safety and efficacy of the biological product.