Highly pure neurotoxic component of a botulinum toxin, process for preparing same, and uses thereof

Patent metadata

Field	Value
Publication number	US-9937245-B2
Application number	US-201414908235-A
Country	US
Kind code	B2
Filing date	Jul 30, 2014
Priority date	Jul 30, 2013
Publication date	Apr 10, 2018
Grant date	Apr 10, 2018

Abstract

The present invention relates to a process for preparing a highly pure neurotoxic component of a botulinum toxin by cultivating clostridium botulinum under conditions that allow production of a botulinum toxin, and isolating the neurotoxic component from the botulinum toxin. In addition, the present invention relates to a highly pure neurotoxic component of a botulinum toxin obtainable by the process of the present invention and uses thereof.

First claim

The invention claimed is:

1. A highly pure neurotoxic component of a Clostridium botulinum toxin having a single-chain content of less than 2.00 wt. %, obtainable by a process comprising:
(a) cultivating Clostridium botulinum under conditions that allow production of a botulinum toxin, and
(b) isolating the neurotoxic component from the botulinum toxin,
wherein cultivating (a) and isolating (b) are conducted in a pressure gradient device, the pressure gradient device comprising a first isolator unit containing a fermenter for cultivating Clostridium botulinum and a safety work bench, and optionally a second or further isolator unit, wherein the first and second or further isolator units are located in a production room that is connected to the environment via an air lock, the pressure in the first and second or further isolator units being lower than that in the production room, the pressure in the production room being lower than ambient pressure, and the pressure in the air lock being higher than ambient pressure, and wherein the safety work bench is located in the production room and is a Class II BSC (Biological Safety Cabinet) provided with a transfer system that allows for the aseptic transfer of material into and out of the BSC.

2. The highly pure neurotoxic component of claim 1 , wherein cultivating temperature is maintained at a temperature of 33.5° C.±1.0° C.

3. The highly pure neurotoxic component of claim 1 , wherein cell density during cultivating (a) is monitored by turbidity measurements using formazin as a primary turbidity standard.

4. The highly pure neurotoxic component of claim 1 , wherein cultivating Clostridium botulinum is continued until cell density decreases from a cell density of 1.3±0.3 FTU reached after 24 hours of cultivation to less than 0.8 FTU.

5. The highly pure neurotoxic component of claim 1 , wherein Clostridium botulinum culture used in (a) is obtained by (i) providing an initial culture of Clostridium botulinum having a cell density of 530 to 850 FTU and (ii) adding a pre-determined amount of initial culture to a fermentation medium.

6. The highly pure neurotoxic component of claim 5 , wherein the initial culture is added to the fermentation medium in an amount of from 5.0% to 10.0% v/v.

7. The highly pure neurotoxic component of claim 1 , wherein the transfer system of the Class II BSC comprises a first transfer unit and a second transfer unit detachably connectable to each other, the first transfer unit being a sealable part mounted on a surface, or fixed to a wall, of the BSC, and the second transfer unit being a sealable container.

8. The highly pure neurotoxic component of claim 1 , wherein the Clostridium botulinum is Clostridium botulinum type A, including the Hall strain (ATCC 3502).

9. The highly pure neurotoxic component of claim 1 , further having a total purity of at least 99.90 wt. %.

10. The highly pure neurotoxic component of claim 1 , further having an endotoxin content of 5.0 IU/ml or less and/or a total anaerobic viable count of less than 1 cfu/ml.

11. A pharmaceutical composition comprising a highly pure neurotoxic component of a Clostridium botulinum toxin according to claim 1 and one or more pharmaceutically acceptable carriers.

12. A medicament comprising a highly pure neurotoxic component of a Clostridium botulinum toxin according to claim 1 .

13. A method for treatment of a disease or condition associated with hyperactive cholinergic innervation of muscles or exocrine glands comprising employing a highly pure neurotoxic component of a Clostridium botulinum toxin according to claim 1 .

14. A method of making the highly pure neurotoxic component of claim 1 , comprising:
(a) cultivating Clostridium botulinum under conditions that allow production of a botulinum toxin, and
(b) isolating the neurotoxic component from the botulinum toxin,
wherein cultivating (a) and isolating (b) are conducted in a pressure gradient device, the pressure gradient device comprising a first isolator unit containing a fermenter for cultivating Clostridium botulinum and a safety work bench, and optionally a second or further isolator unit, wherein the first and second or further isolator units are located in a production room that is connected to the environment via an air lock, the pressure in the first and second or further isolator units being lower than that in the production room, the pressure in the production room being lower than ambient pressure, and the pressure in the air lock being higher than ambient pressure, and wherein the safety work bench is located in the production room and is a Class II BSC (Biological Safety Cabinet) provided with a transfer system that allows for the aseptic transfer of material into and out of the BSC.

15. The method according to claim 14 , wherein cultivating temperature is maintained at a temperature of 33.5° C.±1.0° C.

16. The method according to claim 14 , wherein cell density during cultivating (a) is monitored by turbidity measurements using formazin as a primary turbidity standard.

17. The method according to claim 14 , wherein cultivating Clostridium botulinum is continued until cell density decreases from a cell density of 1.3±0.3 FTU reached after 24 hours of cultivation to less than 0.8 FTU.

18. The method according to claim 14 , further comprising in (a):
(i) providing an initial culture of Clostridium botulinum having a cell density of 530 to 850 FTU and
(ii) adding a pre-determined amount of initial culture to a fermentation medium.

19. The method according to claim 18 , wherein initial culture is added to the fermentation medium in an amount of from 5.0% to 10.0% v/v.

20. The method according to claim 14 , wherein the highly pure neurotoxic component of claim has a total purity of at least 99.90 wt. %.

Assignees

• Merz Pharma Gmbh & Co Kgaa

Inventors

• Pfeil Michael
• Wiesenburg Andreas

Classifications

• A61P25/02

  for peripheral neuropathies · CPC title

• A61P21/00

  Drugs for disorders of the muscular or neuromuscular system · CPC title

• C12N1/20

  Bacteria; Culture media therefor · CPC title

• C12N9/52

  derived from bacteria {or Archaea} · CPC title

• C12M37/00

  Means for sterilizing, maintaining sterile conditions or avoiding chemical or biological contamination (C12M23/38 takes precedence; filtration in general and filters per se B01D24/00-B01D41/00; autoclaves B01J3/04; treatment of microorganisms with electrical or wave energy C12N13/00) · CPC title