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 - 10 . (canceled)
11 . A composition, comprising isolated and purified collagenase I and collagenase II from Clostridium histolyticum, wherein the collagenase I and the collagenase II are substantially clostripain-free and at least 95% pure by area as measured by reverse phase high pressure liquid chromatography (RP-HPLC), wherein the composition is made by a process comprising:
growing Clostridium histolyticum in a medium comprising:
a peptone;
yeast extract; and
greater than about 5 g/L of one or more salts; and
purifying the collagenase I and the collagenase II from the Clostridium histolyticum.
12 . The composition of claim 11 , wherein the medium comprises greater than about 7.5 g/L of the one or more salts.
13 . The composition of claim 12 , wherein the medium comprises greater than about 9 g/L of the one or more salts.
14 . The composition of claim 11 , wherein the one or more salts comprise potassium phosphate, dipotassium phosphate, sodium phosphate, disodium phosphate, sodium chloride, potassium chloride, calcium chloride, or magnesium sulfate.
15 . The composition of claim 13 , wherein the one or more salts comprise potassium phosphate, dipotassium phosphate, disodium phosphate, and sodium chloride.
16 . The composition of claim 15 , wherein the peptone is present at a concentration of about 5% weight by volume to about 15% weight by volume.
17 . The composition of claim 16 , wherein the peptone is present at a concentration of about 10% weight by volume.
18 . The composition of claim 11 , further comprising ferrous sulfate, riboflavin, niacin, calcium pantothenate, pimelic acid, pyridoxine hydrochloride, thiamine hydrochloride, or combinations thereof.
19 . The composition of claim 11 , wherein the purifying comprises:
ammonium sulfate precipitation;
dialysis;
hydroxylapatite chromatography;
gel filtration; and
anion exchange chromatography.
20 . The composition of claim 11 , wherein clostripain production is reduced compared to an amount of clostripain produced from a process that uses a medium comprising less than about 5 g/L of salt.