Methods and Systems for Purifying Non-Complexed Botulinum Neurotoxin
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.
1 . A method for purifying a non-complexed botulinum toxin, the method comprising:
(i) providing a crude non-complexed botulinum toxin;
(ii) loading the crude non-complexed botulinum toxin on an anion exchange column so as to permit capture of the non-complexed botulinum toxin by the anion exchange column;
(iii) eluting the non-complexed botulinum toxin from the anion exchange column to give an eluent comprising the non-complexed botulinum toxin;
(iv) loading a cation exchange column with the eluent from the anion exchange column so as to permit capture of the non-complexed botulinum toxin by the cation exchange column; and
(v) eluting purified non-complexed botulinum toxin from the cation exchange column.
2 . The method according to claim 1 , wherein the crude non-complexed botulinum toxin is obtained by
obtaining a sample comprising botulinum toxin complex;
loading a hydrophobic interaction column with the sample so as to permit capture of the botulinum toxin complex by the hydrophobic interaction column;
eluting the botulinum toxin complex from the hydrophobic interaction chromatography column; and
dissociating the botulinum toxin complex to obtain a mixture comprising the crude non-complexed botulinum toxin.
3 . The method according to claim 2 , wherein the sample is a supernatant or filtrate comprising the botulinum toxin complex.
4 . The method according to claim 2 , wherein the sample is obtained by:
subjecting a fermentation culture comprising the botulinum toxin to acid precipitation to obtain an acid precipitate; and
performing tangential flow filtration on the precipitate to concentrate precipitate.
5 . The method according to claim 2 , wherein the sample is obtained by subjecting an insoluble fraction of a fermentation culture to tangential flow filtration.
6 . The method according to claim 2 , wherein the sample is subjected to a nuclease digestion before loading on the hydrophobic interaction column.
7 . The method according to claim 6 , wherein the nuclease is derived from an animal product free process.
8 . The method according to claim 1 , wherein the method is substantially animal product free.
9 . The method according to claim 1 , wherein the purified non-complexed botulinum toxin comprises at least one of botulinum toxin type A, B, C 1 , D, F, F and G.
10 . The method according to claim 1 , wherein the purified non-complexed botulinum toxin comprises a botulinum toxin type A.
11 . The method according to claim 1 , wherein the purified non-complexed botulinum toxin is at least 95% pure.
12 . The method according to claim 1 , wherein the purified non-complexed botulinum toxin has an activity of at least 200 LD 50 units/ng.
13 . The method according to claim 1 , wherein the method produces a yield of at least about 2 mg/L fermentation culture.
14 . The method according to claim 1 wherein the anionic column is selected from the group consisting of a Q Sepharose HP, Q Sepharose Fast Flow, and Q XL Sepharose column, and wherein the cationic column is selected from the group consisting of a SP Sepharose, SP Sepharose HP, SP Sephrose Fast Flow, Mono S, Source-S, Source-30S, and Source-15S column.
15 . The method according to claim 1 wherein a buffer for loading the non-complexed botulinum toxin onto the anionic column is selected from the group consisting of Tris, bis-Tris, triethanolamine, and N-methyl diethanolamine.
16 . The method according to claim 15 wherein the buffer is used at a pH from 7.4 to 8.2.
17 . The method according to claim 1 wherein a buffer for loading the non-complexed botulinum toxin onto the cationic column is selected from the group consisting of sodium phosphate, MES, and HEPES.
18 . The method according to claim 17 wherein the buffer is used at a pH from 6.0 to 7.0
19 . The method according to claim 1 wherein pH of the anionic column is from 7.4 to 8.2.
20 . The method according to claim 1 wherein pH of the cationic column is from 6.0 to 7.0.
21 . The method according to claim 1 wherein a gradient for eluting the non-complexed botulinum toxin from the anionic column is selected from the group consisting of an ascending gradient of sodium chloride and an ascending gradient of potassium chloride.
22 . The method according to claim 21 wherein the gradient is used at a pH from 7.4 to 8.4.
23 . The method according to claim 1 wherein a gradient for eluting the non-complexed botulinum toxin from the cationic column is selected from the group consisting of an ascending gradient of sodium chloride and an ascending gradient of potassium chloride.
24 . The method according to claim 23 wherein the gradient is used at a pH from 6.0 to 7.0.