Validação de método por eletroforese capilar para avaliação de filgrastima. Estudos de correlação entre métodos físico-químicos e biológico.

Abstract

The granulocyte-colony stimulating factor (G-CSF) is a hematopoietic cytokine that stimulates and regulates the proliferation and differentiation of neutrophils precursors cells of the bone marrow. The recombinant hormone (rhG-CSF) non-glycosylated, filgrastim, is used to treat the neutropenia induced by chemotherapy and bone marrow transplantation. The hydrophobic protein is a 175 aminoacids chain which contains an extra methionine at its N-terminus, and molecular weight of 18.8 kDa. In the present study, a capillary zone electrophoresis (CZE) method was developed and validated for the analysis of filgrastim in pharmaceuticals. The analyses were performed on a fused-silica capillary (75 μm i.d.; effective length, 72 cm) and background electrolyte consisted of 50 mM sodium tetraborate solution at pH 9.0. The capillary temperature was maintained at 15 °C and the applied voltage was 15 kV. The injection was performed using the hydrodynamic mode at 50 mbar for 6 s, with detection at 195 nm using a PDA detector. The electrophoretic separation was obtained with migration time of 21.8 and 15.8 minutes for the filgrastim and leuprorrelin acetate (internal standard), respectively, and with run time of 30 minutes. The procedure was validated by the parameters of specificity, linearity, precision, accuracy, robustness, limit of quantitation and limit of detection. The method was linear in the concentration range of 1 200 μg/mL (r2 = 0.9978) and the limit of quantitation (LOQ) was 1 μg/mL, with acceptable validation parameters. The method was applied for the analysis of pharmaceutical formulations, and the results were correlated to the reversed-phase HPLC method (RP-HPLC), size-exclusion HPLC method (SE-HPLC) and in vitro bioassay method. Therefore, the procedures represent valid alternatives which can improve the quality control, assuring the safety and therapeutic efficacy of the biological product.