Stabilization of thermolysin in aqueous solution
AssigneeRoche Diagnostics Operations, Inc.
InventorsWerner Hoelke, Johann-Peter Thalhofer, Antje Liehre, Markus Weber
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Abstract
The present invention deals with the proteolytic enzyme thermolysin which tends to be unstable in aqueous solution. The invention provides methods and compositions to enhance the stability of dissolved thermolysin in aqueous solution. Thermolysin, crude thermolysin or a lyophilisate containing thermolysin and one or more salts, is contacted with an aqueous buffer with a low salt concentration and a first solution is formed. Subsequently, a further salt in solid form is added and dissociated, thereby forming a second solution comprising thermolysin in a stabilized form.
Claims14
A method for preparing a solution of stabilized thermolysin (EC 3.4.24.27), the method comprising
- the first step (P) of mixing a solid preparation comprising thermolysin with an aqueous solvent and making a first solution, wherein the first solution comprises a buffer salt capable of maintaining a pH in the range of 4.5 to 9, one or more salts, and thermolysin, and in the first solution the aggregate concentration of the one or more salts including the buffer salt(s) is in the range of about 0.1 mM to 500 mM, and
- the subsequent step (Q) of adding to the first solution a measured amount of a further salt, whereby the salt is selected from the group consisting of NaCl, NaBr, NaNO3, NaJ, KCl, LiCl, MgCl2, CaCl2, and a mixture thereof, and dissolving the further salt in the first solution, thereby obtaining a second solution in which the dissolved thermolysin is in a stabilized form.
A liquid composition comprising water, thermolysin in a dissociated form, a dissociated buffer salt capable of maintaining a pH in the range of pH 4.5 and pH 9, and dissociated NaCl at a concentration below 500 mM, and wherein the composition contains thermolysin at a concentration in the range of about 1 mg/ml to about 10 mg/ml.
Dependencies are inferred from the claim text.