# US 10,119,131 B2 · Compositions and methods for producing clostridial collagenases

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- Number: US 10,119,131 B2
- Document ID: US10119131B2
- Status: Granted
- Kind: B2
- Application number: 13/422,939
- Filed: 2012-03-16
- Published: 2012-09-20
- Granted: 2018-11-06
- Earliest claimed: 2011-03-16
- Assignees: BioSpecifics Technologies Corp.
- Applicants: Thomas L. Wegman; Bo Yu
- Inventors: Thomas L. Wegman; Bo Yu
- Web page: https://patentgrep.com/patent/US10119131B2

## CPC

- C12N 9/52: derived from bacteria {or Archaea}
- A61K 38/4886: {Metalloendopeptidases (3.4.24), e.g. collagenase}
- A61P 17/02: for treating wounds, ulcers, burns, scars, keloids, or the like
- A61P 17/10: Anti-acne agents
- A61P 19/04: for non-specific disorders of the connective tissue
- A61P 27/06: Antiglaucoma agents or miotics
- A61P 35/00: Antineoplastic agents
- C12N 1/20: Bacteria; Culture media therefor
- C12Y 304/24003: Microbial collagenase (3.4.24.3)
- C12N 1/00: Microorganisms; Compositions thereof (medicinal preparations containing material from protozoa, bacteria or viruses A61K35/66, from algae A61K36/02, from fungi A61K36/06; preparing medicinal bacterial antigen or antibody compositions, e.g. bacterial vaccines, A61K39/00); Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12P 21/06: produced by the hydrolysis of a peptide bond, e.g. hydrolysate products (preparing foodstuffs by protein hydrolysis A23J3/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12Y: ENZYMES
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}

## 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%.

## Claims

### Claim 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.

### Claim 2 (depends on 1)

The method of claim 1, wherein the medium comprises greater than about 5 g/L total salt.

### Claim 3 (depends on 2)

The method of claim 2, wherein the salt content in the medium is greater than about 7.5 g/L.

### Claim 4 (depends on 1)

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.

### Claim 5 (depends on 4)

The method of claim 4, wherein the optimized fixed mass ratio of collagenase I to collagenase II is 1.

### Claim 6 (depends on 1)

The method of claim 1, wherein the collagenases are 97% pure.

### Claim 7 (depends on 1)

The method of claim 1, wherein the collagenases are 98% pure.

### Claim 8 (depends on 1)

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.

### Claim 9 (depends on 8)

The method of claim 8, wherein steps (i), (ii), (iii), (iv) and (v) are performed in order.

### Claim 10 (depends on 1)

The method of claim 1, wherein the clostripain-producing Clostridium histolyticum is ATCC 21000.

### Claim 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.

### Claim 12 (depends on 11)

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.

### Claim 13 (depends on 12)

The method of claim 12, wherein the optimized fixed mass ratio of collagenase I to collagenase II is 1.

### Claim 14 (depends on 11)

The method of claim 11, wherein the collagenases are 97% pure.

### Claim 15 (depends on 14)

The method of claim 14, wherein the collagenases are 98% pure.

### Claim 16 (depends on 11)

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.

### Claim 17 (depends on 16)

The method of claim 16, wherein steps (i), (ii), (iii), (iv) and (v) are performed in order.

### Claim 18 (depends on 11)

The method of claim 11, wherein the salt content in the medium is greater than about 7.5 g/L.

### Claim 19 (depends on 11)

The method of claim 11, wherein the clostripain-producing Clostridium histolyticum is ATCC 21000.