Avaliação de potência de interferon beta -1b por métodos cromatográficos validados e bioensaio

Abstract

Interferon is a cytokine with antiviral, antiproliferative and immunomodulatory properties that influences cell metabolism, growth and differentiation. The recombinant human interferon β 1b (rhIFNβ-1b) is a non-glycosylated protein produced by the expression in Escherichia coli by the recombinant DNA technology. Structurally, it is a polypeptide chain containing 165 amino acids, molecular weight of 18.5 kDa and isoelectric point of 9.2. The rhIFNβ-1b is clinically used to treat multiple sclerosis. In the present work, reversed-phase liquid chromatography (RP-LC) and size exclusion liquid chromatography (SE-LC) methods were developed and validated for the evaluation of rhIFNβ-1b in biopharmaceutical formulations. The gradient RP-LC method was carried out on a Jupiter C4 column (250 mm x 4.6 mm i.d.) maintained at 30°C. The mobile phase A consisted of 0.1% (v/v) trifluoroacetic acid (TFA) in water and the mobile phase B was 0.1% (v/v) TFA in acetonitrile, run at a flow rate of 1.0 mL/min. The SE-LC method was carried out on a BioSep-SEC-S 2000 column (300 mm x 7.8 mm i.d.) maintained at 25°C. The mobile phase consisted of 1 mM monobasic potassium phosphate, 8 mM sodium phosphate dibasic and 200 mM sodium chloride buffer pH 7.4, run isocratically at a flow rate of 0.8 mL/min. Chromatographic separations were obtained with retention times of 31.87 and 17.78 min, and calibration curves were linear over the concentration range of 1-200 μg/mL (0.032 – 6.4 MIU/mL) (r2 = 0.9998) and 0.50-200 μg/mL (0.016 – 6.4 MIU/mL) (r2 = 0.9999), respectively, for RP-LC and SE-LC, with photodiode array (PDA) detection at 214 nm. The limits of detection and quantitation were 0.47 and 1.57 μg/mL, respectively, for the RP-LC and 0.10 and 0.34 μg/mL for the SE-LC. The specificities were established in degradation studies, which also showed that there was no interference from the formulation excipients. Equally, the accuracy was 100.42 and 100.45%, with bias lower than 0.69 and 0.82%, respectively. 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 rhIFNβ-1b in biotechnology-derived products, giving higher mean differences of the estimated content/potencies of 1.61 and 1.05% for the RP-LC and SE-LC, related to the in vitro assay. It is concluded that represents a contribution to establish new alternatives to monitor stability, improve quality control and thereby assure therapeutic efficacy of the biotechnology-derived medicine.