Validação de métodos cromatográficos para avaliação de estreptoquinase e correlação com o bioensaio

Abstract

The streptokinase (STK) is a secreted and isolated molecule of β-hemolytic streptococci, consists of 414 amino acids and clinically indicated as a thrombolytic agent for myocardial infarction, deep vein thrombosis, pulmonary embolism, acute or subacute peripheral arterial thrombosis. Reversed-phase liquid chromatography (RP-LC) and size exclusion liquid chromatography (SE-LC) methods were developed and validated for the assessment of STK in biopharmaceutical formulations. A RP-LC method was carried out on a Jupiter C4 column (250 mm x 4.6 mm i.d.), Phenomenex, maintained at 25°C. The mobile phase consisted of 50 mM sodium sulphate buffer pH 7.0 and methanol (90:10, v/v), run isocratically at a flow rate of 0.8 mL/min. The SE-LC method was carried out on a Protein KW-802.5 column (300 mm x 8.0 mm i.d.), Shodex, maintained at 25°C. The mobile phase consisted of 40 mM sodium acetate buffer pH 7.0, run isocratically at a flow rate of 1.0 mL/min. Chromatographic separation was obtained with retention times of 19.3 min, and 14.1 min, and was linear over the concentration range of 85 - 25 000 IU/mL (R2 = 0.9999) and 600 – 8 000 IU/mL (R2 = 0.9991), respectively, for RP-LC and SE-LC, with photodiode array (PDA) detection at 220 nm and 204 nm. The limits of detection and quantification were 26 and 85 IU/mL, respectively, for the RP-LC and 190 and 600 IU/mL, for the SE-LC. Specificity was established in degradation studies, which also showed that there was no interference of the excipients. Equally, the accuracy was 100.24% and 99.88%, with bias lower than 0.99% and 0.62%. The validated methods were applied to the determination of STK, and related and higher molecular weight proteins in biopharmaceutical formulations, giving higher mean differences of the estimated content/potencies of 0.46% and 0.53% for the RP-LC and SE-LC related to the chromogenic bioassay, respectively. It is concluded that represents a contribution to establish new alternatives to monitor stability, quality control and thereby assure therapeutic efficacy of the biological medicine.