Methods and systems for purifying non-complexed botulinum neurotoxin

Patent metadata

Field	Value
Publication number	US-9469849-B2
Application number	US-201514796235-A
Country	US
Kind code	B2
Filing date	Jul 10, 2015
Priority date	Oct 21, 2009
Publication date	Oct 18, 2016
Grant date	Oct 18, 2016

Abstract

Methods and systems for chromatographically purifying a botulinum neurotoxin are provided. These methods and systems allow for efficient purification of a non-complexed form of the botulinum neurotoxin in high purity and yield that can be used as an active ingredient in pharmaceutical preparations.

First claim

What is claimed is:

1. A method for purifying a non-complexed botulinum toxin type A ( botulinum toxin A), the method comprising:
(a) providing a mixture comprising a crude non-complexed botulinum toxin A, in which said crude non-complexed botulinum toxin A is dissociated from native non-toxin proteins; wherein said mixture is obtained by:
(i) subjecting a fermentation culture comprising botulinum toxin A to acid precipitation to produce an insoluble acid precipitate;
(ii) concentrating the acid precipitate from step (i) to produce a concentrated sample;
(iii) subjecting the sample to nuclease digestion under conditions which reduce host cell nucleic acid content and which maintain a complex of botulinum toxin A and non-toxin proteins;
(iv) removing cell debris from the sample of step (iii) to produce a clarified sample;
(v) loading a hydrophobic interaction column with the clarified sample from step (iv) under conditions to permit capture of the botulinum toxin A complex by the hydrophobic interaction column and impurities to flow through the column;
(vi) eluting the botulinum toxin A complex from the hydrophobic interaction column of step (vi); and
(vii) dissociating the botulinum toxin A complex obtained from step (vi) under conditions which disrupt the complex and produce a mixture comprising crude non-complexed botulinum toxin A dissociated from the native non-toxin proteins;

(b) loading the mixture containing the crude non-complexed botulinum toxin A dissociated from native non-toxin proteins from step (a) on an anion exchange column under conditions permitting capture of the non-complexed botulinum toxin A by the anion exchange column;
(c) eluting the non-complexed botulinum toxin A from the anion exchange column of step (b) to give an eluent comprising the non-complexed botulinum toxin A;
(d) loading a cation exchange column with the eluent from the anion exchange column of step (c) under conditions permitting capture of the non-complexed botulinum toxin A by the cation exchange column; and
(e) eluting purified non-complexed botulinum toxin A from the cation exchange column of step (d).

2. The method according to claim 1 , wherein, in step (i), the fermentation culture comprising botulinum toxin A is acid precipitated with sulfuric acid.

3. The method according to claim 2 , wherein the concentrated sample of step (ii) is obtained by performing tangential flow filtration on the acid precipitate to concentrate the precipitate.

4. The method according to claim 1 , wherein the sample of step (iii) is subjected to nuclease digestion at a pH of 5 to 7.

5. The method according to claim 4 , wherein the nuclease is derived from a non-animal source.

6. The method according to claim 5 , wherein, in step (iv), the cell debris is removed by centrifugation and/or filtration to produce the clarified sample.

7. The method according to claim 6 , wherein the clarified sample of step (iv) is combined with a buffer comprising ammonium sulfate prior to loading the hydrophobic interaction column in step (v).

8. The method according to claim 7 , wherein the clarified sample of step (iv) is combined with a buffer comprising ammonium sulfate and phosphate at pH 6 prior to loading the hydrophobic interaction column in step (v).

9. The method according to claim 8 , wherein, in step (vii), the botulinum toxin A complex is dissociated in a buffer having a pH of 7.0 to 8.4 to obtain the mixture comprising the crude non-complexed botulinum toxin A dissociated from the native non-toxin proteins.

10. The method according to claim 9 , wherein the botulinum toxin A complex is dissociated in Tris buffer at pH 7.8.

11. The method according to claim 10 , wherein the method is substantially animal product free.

12. The method according to claim 11 , wherein the hydrophobic interaction column comprises a chromatography gel matrix comprising beaded agarose, having attached thereto chemical groups selected from butyl, octyl, or phenyl groups.

13. The method according to claim 12 , wherein a loading or equilibration buffer for the hydrophobic interaction column is selected from acetate, citrate, 2-(N-morpholino)ethanesulfonic acid (MES), histidine, piperazine and malonate.

14. The method according to claim 13 , wherein the pH of the hydrophobic interaction column buffer is in the range of pH 4.0 to 7.0.

15. The method according to claim 14 , wherein the pH of the hydrophobic interaction column buffer is in the range of pH 4.5 to 6.5.

16. The method according to claim 15 , wherein the pH of the hydrophobic interaction column buffer is pH 6.

17. The method according to claim 12 , wherein the bound botulinum toxin A complex is eluted from the hydrophobic interaction column in a descending gradient of an elution buffer selected from the group consisting of ammonium sulfate, sodium sulfate, sodium chloride, potassium chloride and ammonium acetate.

18. The method according to claim 17 , wherein the elution buffer is ammonium sulfate.

19. The method according to claim 17 , wherein the concentration range of the descending gradient elution buffer is from 0.6 M to 0.0 M.

20. The method according to claim 17 , wherein the concentration range of the descending gradient elution buffer is from 0.5 M to 0.0 M.

21. The method according to claim 17 , wherein the concentration range of the descending gradient elution buffer is from 0.4 M to 0.0 M.

22. The method according to claim 17 , wherein the concentration range of the descending gradient elution buffer is from 0.25 M to 0.0 M.

23. The method according to claim 12 , wherein the bound botulinum toxin A complex is eluted from the hydrophobic interaction column in a descending gradient of ammonium sulfate elution buffer in a concentration range of 0.4 M to 0.0 M at pH 6.

24. The method according to claim 17 , wherein the anion exchange column comprises a chromatography gel matrix comprising beaded agarose having a net negative charge, and wherein the cation exchange column comprises a chromatography gel matrix comprising beaded agarose having a net positive charge; wherein said anion and cation exchange columns provide separation of proteins based on electrostatic charge.

25. The method according to claim 24 , wherein the non-complexed botulinum toxin A is loaded onto the anion exchange column in a buffer selected from the group consisting of Tris, bis-Tris, triethanolamine, and N-methyl diethanolamine.

26. The method according to claim 25 , wherein the buffer is used at a pH from 7.2 to 8.6.

27. The method according to claim 25 , wherein the buffer is used at a pH from 7.4 to 8.2.

28. The method according to claim 24 , wherein the non-complexed botulinum toxin A is loaded onto the cation exchange column in a buffer selected from the group consisting of sodium phosphate, 2-(N-morpholino)ethanesulfonic acid (MES) and HEPES.

29. The method according to claim 28 , wherein the buffer is used at a pH of from 6.0 to 7.0.

30. The method according to claim 24 , wherein the pH of the anion exchange column is from 7.4 to 8.2.

31. The method according to claim 30 , wherein the buffer is used at a pH of from 6.8 to 7.0.

32. The method according to claim 24 , wherein a concentration gradient of an elution buffer for eluting the non-comp

Assignees

• Revance Therapeutics Inc

Inventors

• Ruegg Curtis L

Classifications

• C12N9/52 Primary

  derived from bacteria {or Archaea} · CPC title

• C07K14/33 Primary

  from Clostridium (G) · CPC title

• C12Y304/24069

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