COMPOSITIONS AND METHODS FOR PRODUCING CLOSTRIDIAL COLLAGENASES
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.