Compositions and methods for producing clostridial collagenases
View Patent ↗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 method for fermenting a clostripain-producing Clostridium histolyticum to obtain a mixture of collagenase I and collagenase II, comprising fermenting said clostripain-producing Clostridium histolyticum in medium comprising peptone, wherein the peptone is Oxoid VG100 Vegetable Peptone made from pea, or Oxoid VG200 Vegetable Peptone phosphate broth,
wherein said mixture of collagenase I and collagenase II is separately purified in the absence of added protease inhibitor and recombined to produce a drug product at least 95% pure as determined by reverse phase high performance liquid chromatography and contains no detectable clostripain activity using an enzymatic assay with N-a-Benzoyl-L-Arginine Ethyl Ester (BAEE) as substrate.
2. The method of claim 1 , wherein the medium comprises greater than about 5 g/L total salt.
3. The method of claim 2 , wherein the salt content in the medium is greater than about 7.5 g/L.
4. The method of claim 1 , wherein the collagenase I and collagenase II are recombined at an optimized fixed mass ratio of 0.5 to 1.5.
5. The method of claim 4 , wherein the optimized fixed mass ratio of collagenase I to collagenase II is 1.
6. The method of claim 1 , wherein the collagenases are 97% pure.
7. The method of claim 1 , wherein the collagenases are 98% pure.
8. The method of claim 1 , wherein the purifying comprises:
i. ammonium sulfate precipitation;
ii. dialysis;
iii. hydroxylapatite chromatography;
iv. gel filtration; and
v. anion exchange.
9. The method of claim 8 , wherein steps (i), (ii), (iii), (iv) and (v) are performed in order.
10. The method of claim 1 , wherein the clostripain-producing Clostridium histolyticum is ATCC 21000.
11. A method for fermenting a clostripain-producing Clostridium histolyticum to obtain a mixture of collagenase I and collagenase II, comprising fermenting said clostripain-producing Clostridium histolyticum in medium comprising peptone and lacking glucose, wherein the peptone is BBL Phytone Peptone, or BD Difco Select Phytone,
wherein said mixture of collagenase I and collagenase II is separately purified in the absence of added protease inhibitor and recombined to produce a drug product at least 95% pure as determined by reverse phase high performance liquid chromatography and contains no detectable clostripain activity using an enzymatic assay with N-a-Benzoyl-L-Arginine Ethyl Ester (BAEE) as substrate.
12. The method of claim 11 , wherein the collagenase I and collagenase II are recombined at an optimized fixed mass ratio of 0.5 to 1.5.
13. The method of claim 12 , wherein the optimized fixed mass ratio of collagenase I to collagenase II is 1.
14. The method of claim 11 , wherein the collagenases are 97% pure.
15. The method of claim 14 , wherein the collagenases are 98% pure.
16. The method of claim 11 , wherein the purifying comprises:
i. ammonium sulfate precipitation;
ii. dialysis;
iii. hydroxylapatite chromatography;
iv. gel filtration; and
v. anion exchange.
17. The method of claim 16 , wherein steps (i), (ii), (iii), (iv) and (v) are performed in order.
18. The method of claim 11 , wherein the salt content in the medium is greater than about 7.5 g/L.
19. The method of claim 11 , wherein the clostripain-producing Clostridium histolyticum is ATCC 21000.