Formulation of sustained release aqueous Zn-hirudin suspensions

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 ZnCl2 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 (similar to 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 mu m were observed by light microscopy. Zinc salt-induced precipitation phenomena were also investigated using ZnBr2, ZnI2, Zn(NO3)(2) and ZnSO4 instead of ZnCl2. ZnSO4 showed the lowest precipitation efficiency. All other salts behaved similar to ZnCl2. Upon storage the pelletized protein content of the ZnCl2 based precipitates was stable (similar to 95% rHir after 1 year at room temperature), whereas the pelletized protein content of ZnSO4 based precipitates dropped sharply after precipitation (2% remaining after 13 days at room temperature). This indicates a transition of the ZnSO4 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. (C) 2000 Elsevier Science B.V. All rights reserved.