Development of cryopelletization and formulation measures to improve stability of Echis carinatus venum protein for use in diagnostic rotational thromboelastometry.

A cryopellet formulation of the diagnostic protein ecarin has been developed that is suitable for use to monitor blood coagulation via in vitro thromboelastometry. The coagulation activity of the ecarin was measured by thromelastometry using whole blood. In aqueous solution the ecarin is inactivated rapidly in a temperature dependent way that deviates from the Arrhenius equation. When kept at room temperature the ecarin activity falls to 90% of its initial value after just 39 min. The stability kinetics of ecarin during freezing and lyophilization are strongly dependent on the presence of a stabilizer comprising a collagen peptide-fraction. If this is removed by ultra-filtration the ecarin becomes highly unstable and cannot be fully stabilized even by addition of trehalose. The storage stability of ecarin in the finished pellets is excellent at temperatures below 50 °C, but deteriorates above the glass transition temperature of the pellet formulation. The cryopelletization of ecarin offer therefore a stable formulation for use in thromboelastometry that is superior to an aqueous solution and has much better handling than a regular lyophilizate in a diagnostic device.