Urokinase: Stability Studies in Solution and Lyophilized Formulations

Abstract

Urokinase - a serine protease - is used clinically as a thrombolytic agent to dissolve blood clots. Low molecular weight Urokinase (33,000 dalton) isolated from human kidney cells using tissue culture techniques was used in the stability studies. Quantitative determination of Urokinase was accomplished by either amidolytic or fibrinolytic activity assay methods. The degradation of Urokinase in solution at 55 °C follows pseudo-first order kinetics at several pH values. The pH range for maximum stability has been determined to be about 6 to 7.

The stability of Urokinase is very sensitive to the quantity of residual moisture in the lyophilized formulation. Rubber stoppers used as closures for the glass vials were identified as a major source of moisture. The loss of activity from freeze dried formulations was inversely related to the specific activity of tissue culture derived Urokinase. Similar relationship was also observed for the adsorption of Urokinase from 5% dextrose diluent to the surface of polyvinyl chloride large volume parenteral diluent bags. Initial degradation rates (zero order) for freeze dried urokinase formulations with and without the addition of human serum albumin (HSA) as a stabilizer determined at 50, 40 and 30 °C demonstrated that loss of urokinase followed the Arrhenius relationship with an apparent energy of activation (Ea) of 15 kcal per mol. The addition of HSA resulted in an increase in stability by about a factor of four. However, the apparent Ea for the formulations with and without HSA was not significantly different as evident from parallel slopes in the Arrhenius plots.