Formulation of sustained release aqueous Zn–hirudin suspensions

Abstract

Sustained release formulations for recombinant hirudin (rHir), an anticoagulant thrombin-specific inhibitor, were developed. Zn–rHir suspensions were formed by precipitation with zinc salts at neutral pH. Characterization of protein precipitation was by UV analysis, capillary electrophoresis (CE), zinc analysis, light and electron microscopy, and particle size analysis. The precipitation of aqueous rHir solution with ZnCl 2 solution at neutral pH resulted in Zn–rHir suspensions. Optimum yields of pelletized Zn–rHir were obtained between pH 7.0 and 7.4. For complete precipitation (∼100%) a molar ratio of zinc to rHir of >28 was necessary. As shown by electron microscopy, the smallest resolvable unit of Zn–rHir suspensions was 20 nm. Agglomerates of up to 200 μm were observed by light microscopy. Zinc salt-induced precipitation phenomena were also investigated using ZnBr 2, ZnI 2, Zn(NO 3) 2 and ZnSO 4 instead of ZnCl 2. ZnSO 4 showed the lowest precipitation efficiency. All other salts behaved similar to ZnCl 2. Upon storage the pelletized protein content of the ZnCl 2 based precipitates was stable (∼95% rHir after 1 year at room temperature), whereas the pelletized protein content of ZnSO 4 based precipitates dropped sharply after precipitation (2% remaining after 13 days at room temperature). This indicates a transition of the ZnSO 4 based precipitates to hexagonal basic zinc sulfate plates and free rHir. The driving force is the lower aqueous solubility of basic zinc sulfate as compared to the higher solubility of basic zinc chloride.