Estudos analíticos para caracterização e avaliação de interleucina-11 humana recombinante

Abstract

Interleukin 11 (IL-11) is an endogenous cytokine that directly stimulates the proliferation of hematopoietic stem cells and megakaryocytic progenitor cells and induces megakaryocytic maturation. With advances in biotechnology, particularly with the advent of recombinant DNA technology has been enabled the expression of the IL-11 gene in Escherichia coli and thus the large-scale production of interleukin-11 Recombinant human (rhIL-11), which is clinically indicated, especially for severe thrombocytopenia to prevent and reduce the need for platelet transfusions following myelosuppressive chemotherapy in patients with non-myeloid malignancies at high risk of serious trompocitopenia. The analysis for CZE method were performed on a fused-silica capillary (effective length, 40 cm; 50 μm i.d.), using electrolyte solution consisted of 50 mM dihydrogen phospate solution at pH 3.0. The capillary was maintained at 25º C, the applied voltage was 20 kV. Injections were performed using a pressure mode at 50 mbar for 45 s, with detection by photodiode array detector set at 196 nm. The SE-LC method was carried out on a BioSep-SEC-S 2000 column (300 mm x 7.8 mm i.d.), maintained at 30ºC. The mobile phase consisted of 0.02 M 2-(N-morpholino)ethanesulfonic acid and 0.5 M sodium chloride buffer, pH 6.0, run isocratically at a flow rate of 0.9 mL/min, and using a photodiode array (PDA) detection at 220 nm. Chromatographic separation was obtained with retention times of 10.31 min, and 8.12 min, and was linear over the concentration range of 1-300 μg/mL (r2 = 0.9992) and 1-200 μg/mL (r2 = 0.9996), respectively, for CZE and SE-LC. The limits of detection and quantitation were 0.21 and 1.0 μg/mL, respectively, for the CZE and 0.23 and 1.0 μg/mL, for the SE-LC. The specificity of the methods has been verified and confirmed by studies of degradation or interference of the excipients. Equally, the accuracy was 100.37% and 99.81%, with bias lower than 1.13% and than 0.43%, respectively, for CZE and SE-LC. Moreover, the in vitro cytotoxicity test of related proteins and higher molecular weight forms showed significant differences (p< 0.05) compared to intact molecule. The validated methods were applied for the determination of rhIL-11 and related proteins in biotechnology-derived products, and the results were correlated to those of a validated reversed-phase LC method (RP-LC) and an TF-1 cell culture assay, with significant correlation (p> 0.05). Thus, we suggest the application of methods developed and validated by CZE and SE-LC to improve quality control of rhIL-11 biotechnology-derived product, thereby contributing to ensure their safety and therapeutic efficacy.