Process for preparing a highly pure neurotoxic component of a botulinum toxin and uses thereof

Patent metadata

Field	Value
Publication number	US-2016175407-A1
Application number	US-201414908235-A
Country	US
Kind code	A1
Filing date	Jul 30, 2014
Priority date	Jul 30, 2013
Publication date	Jun 23, 2016
Grant date	—

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 com-ponent 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

1 . A process for preparing a highly pure neurotoxic component of a botulinum toxin, 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, which comprises a first isolator unit containing a fermenter for cultivating Clostridium botulinum and, optionally, a second or further isolator unit, the first and second or further isolator units being located in a production room that is connected to the environment via an air lock, wherein the pressure in the first and second or further isolator units is lower than that in the production room, the pressure in the production room is lower than ambient pressure, and the pressure in the air lok is higher than ambient pressure, and wherein the pressure gradient device further comprises a safety work bench which 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, the safety work bench being also located in the production room.

2 . The process of claim 1 , wherein the cultivating temperature is maintained at a temperature of 33.5° C.±1.0° C.

3 . The process of claim 1 , wherein the cell density during cultivation
(a) is monitored by turbidity measurements using formazin as a primary turbidity standard.

4 . The process of claim 1 , wherein the cultivation of 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 process of claim 1 , wherein the 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 process 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 process 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 process of claim 1 , wherein the Clostridium botulinum is Clostridium botulinum type A, including the Hall strain (ATCC 3502).

9 . A highly pure neurotoxic component of a Clostridium botulinum toxin having a single-chain content of less than 2.00 wt. %.

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

11 . The highly pure neurotoxic component of claim 9 , 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.

12 . The highly pure neurotoxic component of any one of claim 9 , obtainable by the process according to claim 1 .

13 . A pharmaceutical composition comprising a highly pure neurotoxic component of a Clostridium botulinum toxin according to claim 9 and one or more pharmaceutically acceptable carriers.

14 . A highly pure neurotoxic component of a Clostridium botulinum toxin according to claim 9 for use as a medicament.

15 . A highly pure neurotoxic component of a Clostridium botulinum toxin according to claim 9 for use in the treatment of a disease or condition associated with hyperactive cholinergic innervation of muscles or exocrine glands.

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

• C12N9/52

  derived from bacteria {or Archaea} · CPC title

• C07K14/33

  from Clostridium (G) · CPC title

• C12Y304/24069

  Bontoxilysin (3.4.24.69), i.e. botulinum neurotoxin · CPC title