Roche Diagnostics Operations, Inc.

Roche Diagnostics Gmbh

F. Hoffmann-la Roche Ag

Michaela Fischer

Johann-Peter Thalhofer

Werner Hoelke

Bernhard Suppmann

Antje Liehre

Markus Weber

Hellmut Eckstein

Described are compositions, in particular lyophilizates, containing proteolytic enzymes, and methods for producing the compositions. Typically these compositions contain one or more proteases with collagenase activity and a neutral protease, for example, thermolysin. The compositions are free of acetate salts. Surprisingly, such compositions can be dissolved in water more rapidly than lyophilized protease mixtures of the state of the art.

Methods of preparing a solid composition containing proteases

The present invention provides a method for purifyingcollagenase type I and type II proteins from a complex mixture by subsequently performing a precipitation with ammonium sulfate, hydrophobic interaction chromatography, cation exchange chromatography, and anion chromatography. Conditions are provided which lead to a stabilized, partially purified preparation even after the precipitation step. The method of the invention leads to a quick and efficient removal of other proteolytic activities. The preparations according to the invention provide exceptionally pure and intact collagenase type I and type II proteins which are enzymatically active. The invention also provides blends of the two isolated proteins. The invention further provides the use of the purified collagenase proteins or blends thereof for treating a tissue sample in vitro.

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Wolfratshausen, Germany

Michaela Fischer