Methods and Systems for Purifying Non-Complexed Botulinum Neurotoxin

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.

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.