Compositions and methods for producing clostridial collagenases

Abstract

The present invention provides a method for producing a drug product comprising a combination of highly purified collagenase I and collagenase II from Clostridium histolyticum. The method utilizes an improved medium for the cultivation of Clostridium histolyticum which includes a non-meat-derived (i.e., non-mammalian) peptone or vegetable peptone. The method includes one or more of: (1) reducing glucose content in the meat-free or vegetable-derived media; and (2) increasing the salt concentration in the meat-free or vegetable-derived media. Also provided is a drug product which includes collagenase I and collagenase II at an optimized fixed mass ratio, and which has a purity of greater than at least 95%.

1 . A medium for the fermentation of  Clostridium histolyticum comprising:    a. a peptone selected from the group consisting of Oxoid VG100 Vegetable Peptone made from pea, Oxoid VG200 Vegetable Peptone phosphate broth, BBL Phytone Peptone, and BD Difco Select Phytone;   b. yeast extract; and   c. less than about 5 g/L glucose;   wherein the pH of said medium is 7.5-7.9.   
     
     
         2 . The medium of  claim 1 , containing greater than about 5 g/L salt. 
     
     
         3 . The medium of  claim 2 , containing greater than about 7.5 g/L salt. 
     
     
         4 . The medium of  claim 2 , wherein the salt is one or more of potassium phosphate, dipotassium phosphate, sodium phosphate, disodium phosphate, sodium chloride, potassium chloride, calcium chloride, or magnesium sulfate. 
     
     
         5 . The medium of  claim 1 , further comprising one or more vitamins selected from the group consisting of ferrous sulfate, riboflavin, niacin, calcium pantothenate, pimelic acid, pyridoxine hydrochloride and thiamine hydrochloride. 
     
     
         6 . A medium for the fermentation of  Clostridium histolyticum  comprising:
 a. a peptone selected from the group consisting of Oxoid VG100 Vegetable Peptone made from pea, Oxoid VG200 Vegetable Peptone phosphate broth, BBL Phytone Peptone, and BD Difco Select Phytone;   b. yeast extract; and   c. greater than about 5 g/L salt;   wherein the pH of said medium is 7.5-7.9.   
     
     
         7 . The medium of  claim 6 , containing less than about 5 g/L glucose. 
     
     
         8 . The medium of  claim 6 , containing greater than about 7.5 g/L salt. 
     
     
         9 . The medium of  claim 6 , wherein the salt is one or more of potassium phosphate, dipotassium phosphate, sodium phosphate, disodium phosphate, sodium chloride, potassium chloride, calcium chloride, or magnesium sulfate. 
     
     
         10 . The medium of  claim 6 , further comprising one or more vitamins selected from the group consisting of ferrous sulfate, riboflavin, niacin, calcium pantothenate, pimelic acid, pyridoxine hydrochloride and thiamine hydrochloride.