Methods for purifying clostridial neurotoxin
A method for purifying a clostridial neurotoxin comprising contacting a cation exchange resin with a composition comprising a clostridial neurotoxin, wherein the contacting step is performed at at least pH 7.3, wherein the step of contacting a cation exchange resin with a composition comprising said clostridial neurotoxin occurs prior to conversion of the clostridial neurotoxin from a single chain form into a dichain form. Also provided are uses of a buffer having a pH value that is −1 pH unit or higher than the calculated pi of a clostridial neurotoxin, purification intermediates and clostridial neurotoxins obtainable by the invention, wherein the clostridial neurotoxin is in a single chain form.
1. A method of producing the active di-chain form of a clostridial neurotoxin, the method comprising:
(a) contacting a cation exchange resin with a composition comprising a single-chain clostridial neurotoxin, wherein the contacting step is performed at a pH of from 7.3 to 9.5;
(b) separating the clostridial neurotoxin from the cation exchange resin and
(c) contacting the separated clostridial neurotoxin with a protease that cleaves the polypeptide sequence of the clostridial neurotoxin under conditions suitable for the cleavage, thereby activating it to produce the active di-chain form of the clostridial neurotoxin,
wherein the step (a) occurs before the step (b).
2. The method of claim 1 , wherein the separated clostridial neurotoxin is in a substantially pure state.
3. The method of claim 1 , wherein the contacting step is performed at a pH of from 7.5 to 9.0.
4. The method of claim 1 , wherein the contacting step is performed at a pH of from 7.5 to 8.5.
5. The method of claim 1 , wherein the contacting step is performed at a pH of from 7.3 to 8.0.
6. The method of claim 1 , wherein the contacting step is performed in a buffer having a pH that is −1 pH unit or higher than the calculated pI of the clostridial neurotoxin.
7. The method of claim 6 , wherein the buffer has a pH value that is equal to or higher than the calculated pI of said clostridial neurotoxin.
8. The method of claim 6 , wherein the buffer has a pH of between 0.2 and 1.5 pH units higher than the calculated pI of said clostridial neurotoxin.
9. The method of claim 6 , wherein the buffer has a pH of between 0.2 and 1.0 pH units higher than the calculated pI of said clostridial neurotoxin.
10. The method of claim 1 , further comprising contacting the composition comprising the single-chain clostridial neurotoxin with one or more additional resins.
11. The method of claim 1 , wherein at least 35% of the total clostridial neurotoxin comprised in the composition associates with the cation exchange resin.
12. The method of claim 1 , wherein the clostridial neurotoxin is a Clostridium botulinum neurotoxin, a Clostridium tetani neurotoxin, a Clostridium baratii neurotoxin, or a C. butyricum neurotoxin.
13. The method of claim 1 , wherein the clostridial neurotoxin is a botulinum neurotoxin (BoNT).
14. The method of claim 13 , wherein the BoNT is a cationic BoNT.
15. The method of claim 1 , wherein the protease is an endoprotease.
16. The method of claim 15 , wherein the endoprotease is Lys-C.