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.

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  or  claim 3 , 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 1 , wherein the hydrophobic interaction column comprises a matrix selected from butyl sepharose, phenyl sepharose, or octyl sepharose. 
     
     
         13 . The method according to  claim 12 , wherein the hydrophobic interaction column is selected from the group consisting of a Butyl Sepharose® hydrophobic interaction column, a Phenyl Sepharose® hydrophobic interaction column, a Phenyl Sepharose® Fast Flow high substitution hydrophobic interaction column, a Phenyl Sepharose® Fast Flow low substitution hydrophobic interaction column and an Octyl Sepharose® hydrophobic interaction column. 
     
     
         14 . The method according to  claim 13 , 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. 
     
     
         15 . The method according to  claim 14 , wherein the pH of the hydrophobic interaction column buffer is in the range of pH 4.0 to 7.0. 
     
     
         16 . The method according to  claim 15 , wherein the pH of the hydrophobic interaction column buffer is in the range of pH 4.5 to 6.5. 
     
     
         17 . The method according to  claim 16 , wherein the pH of the hydrophobic interaction column buffer is pH 6. 
     
     
         18 . The method according to  claim 13 , 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. 
     
     
         19 . The method according to  claim 18 , wherein the elution buffer is ammonium sulfate. 
     
     
         20 . The method according to  claim 18 , wherein the concentration range of the descending gradient elution buffer is from 0.6 M to 0.0 M. 
     
     
         21 . The method according to  claim 18 , wherein the concentration range of the descending gradient elution buffer is from 0.5 M to 0.0 M. 
     
     
         22 . The method according to  claim 18 , wherein the concentration range of the descending gradient elution buffer is from 0.4 M to 0.0 M. 
     
     
         23 . The method according to  claim 18 , wherein the concentration range of the descending gradient elution buffer is from 0.25 M to 0.0 M. 
     
     
         24 . The method according to  claim 13 , 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. 
     
     
         25 . The method according to  claim 18 , wherein the anion exchange column comprises a matrix having a net negative charge and is selected from the group consisting of a Q Sepharose® HP anion exchange column, a Q Sepharose® Fast Flow anion exchange column, and Q XL Sepharose® anion exchange column, and wherein the cation exchange column comprises a matrix having a net positive charge and is selected from the group consisting of a SP Sepharose® cation exchange column, a SP Sepharose® HP cation exchange column, a SP Sepharose® Fast Flow cation exchange column, a Mono S® cation exchange column, a Source-S® cation exchange column, a Source™-30S cation exchange column, and a Source™-15S cation exchange column. 
     
     
         26 . The method according to  claim 25 , 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. 
     
     
         27 . The method according to  claim 26 , wherein the buffer is used at a pH from 7.2 to 8.6. 
     
     
         28 . The method according to  claim 26 , wherein the buffer is used at a pH from 7.4 to 8.2. 
     
     
         29 . The method according to  claim 25 , 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. 
     
     
         30 . The method according to  claim 29 , wherein the buffer is used at a pH from 6.0 to 7.0. 
     
     
         31 . The method according to  claim 25 , wherein the pH of the anion exchange column is from 7.4 to 8.2. 
     
     
         32 . The method according to  claim 31 , wherein pH of the cation exchange column is from 6.8 to 7.0. 
     
     
         33 . The method according to  claim 25 , wherein a concentration gradient of an elution buffer for eluting the non-complexed  botulinum  toxin A from the anion exchange column is selected from the group consisting of an ascending concentration gradient of sodium chloride and an ascending concentration gradient of potassium chloride. 
     
     
         34 . The method according to  claim 33 , wherein the concentration range of the ascending gradient is from 0.0 M to 0.6 M. 
     
     
         35 . The method according to  claim 33 , wherein concentration range of the ascending gradient is from 0.0 M to 0.5 M. 
     
     
         36 . The method according to  claim 33 , wherein the ascending concentration gradient of sodium chloride has a concentration range of 0.0 M to 0.6 M. 
     
     
         37 . The method according to  claim 33 , wherein the anion exchange column is eluted at a pH from 7.4 to 8.4. 
     
     
         38 . The method according to  claim 25 , wherein a gradient for eluting the non-complexed  botulinum  toxin A from the cation exchange column is selected from the group consisting of an ascending concentration gradient of sodium chloride and an ascending concentration gradient of potassium chloride. 
     
     
         39 . The method according to  claim 38 , wherein the concentration range of the ascending gradient of sodium chloride is from 0.0 M to 1 M. 
     
     
         40 . The method according to  claim 38 , wherein concentration range of the ascending gradient of potassium chloride is from 0.0 M to 0.5 M. 
     
     
         41 . The method according to  claim 38 , wherein the cation exchange column is eluted at a pH from 6.0 to 7.0. 
     
     
         42 . The method according to  claim 41 , wherein the pH of the cation exchange column is pH 7. 
     
     
         43 . The method according to  claim 1 , wherein
 the hydrophobic interaction column comprises a matrix selected from butyl sepharose, phenyl sepharose, or octyl sepharose;   the anion exchange column comprises a matrix having a net negative charge and is selected from the group consisting of a Q Sepharose® HP anion exchange column, a Q Sepharose® Fast Flow anion exchange column, and Q XL Sepharose® anion exchange column; and   the cation exchange column comprises a matrix having a net positive charge and is selected from the group consisting of a SP Sepharose® cation exchange column, a SP Sepharose® HP cation exchange column, a SP Sepharose® Fast Flow cation exchange column, a Mono S® cation exchange column, a Source-S® cation exchange column, a Source™-30S cation exchange column, and a Source™-15S cation exchange column.   
     
     
         44 . The method according to  claim 43 , wherein the pH of the hydrophobic interaction column buffer is in the range of pH 4.0 to 7.0. 
     
     
         45 . The method according to  claim 44 , wherein the pH of the hydrophobic interaction column buffer is in the range of pH 5.5 to 6.5. 
     
     
         46 . The method according to  claim 43 , wherein the bound  botulinum  toxin A complex is eluted from the hydrophobic interaction column in an ammonium sulfate buffer in a descending concentration gradient of from 0.6 M. to 0.0 M. 
     
     
         47 . The method according to  claim 43 , wherein the bound  botulinum  toxin A complex is eluted from the hydrophobic interaction column in an ammonium sulfate buffer in a descending concentration gradient of from 0.5 M to 0.0 M. 
     
     
         48 . The method according to  claim 43 , wherein the bound  botulinum  toxin A complex is eluted from the hydrophobic interaction column in an ammonium sulfate buffer in a descending concentration gradient from 0.4 M. to 0.0 M. 
     
     
         49 . The method according to any one of  claims 46  to  48 , wherein the bound  botulinum  toxin A complex is eluted from the hydrophobic interaction column at a pH of 6.0. 
     
     
         50 . The method according to  claim 43 , 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. 
     
     
         51 . The method according to  claim 50 , wherein the pH of the anion exchange column buffer is in the range of pH 7.0 to 8.4. 
     
     
         52 . The method according to  claim 51 , wherein the pH of the anion exchange column buffer is in the range of pH 7.4 to 8.2. 
     
     
         53 . The method according to  claim 43 , 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. 
     
     
         54 . The method according to  claim 53 , wherein the pH of the cation exchange column buffer is in the range of pH 4.0 to 8.0. 
     
     
         55 . The method according to  claim 54 , wherein the pH of the cation exchange column buffer is in the range of pH 5.0 to 7.5. 
     
     
         56 . The method according to  claim 54 , wherein the pH of the cation exchange column buffer is in the range of pH 6.0 to 7.0. 
     
     
         57 . The method according to  claim 43 , wherein the non-complexed  botulinum  toxin A is eluted from the anion exchange column in a buffer selected from sodium chloride or potassium chloride in an ascending concentration gradient of 0.0 M to 0.6 M. 
     
     
         58 . The method according to  claim 57 , wherein the non-complexed  botulinum  toxin A is eluted from the anion exchange column in an ascending concentration gradient of sodium chloride in a concentration range of 0.0 M to 0.4 M. 
     
     
         59 . The method according to  claim 57 , wherein the non-complexed  botulinum  toxin A is eluted from the anion exchange column in an ascending concentration gradient of sodium chloride in a concentration range of 0.0 M to 0.5 M. 
     
     
         60 . The method according to  claim 57 , wherein the non-complexed  botulinum  toxin A is eluted from the anion exchange column in an ascending concentration gradient of sodium chloride in a concentration range of 0.0 M to 0.6 M. 
     
     
         61 . The method according to  claim 57 , wherein the non-complexed  botulinum  toxin A is eluted from the anion exchange column at a pH of 7.4 to 8.4. 
     
     
         62 . The method according  claim 43 , wherein the non-complexed  botulinum  toxin A is eluted from the cation exchange column in an ascending concentration gradient of sodium chloride in a concentration range of from 0.0 M to 1.0 M. 
     
     
         63 . The method according to  claim 43 , wherein the non-complexed  botulinum  toxin A is eluted from the cation exchange column in an ascending concentration gradient of potassium chloride in a concentration range of from 0.0 M to 0.5 M. 
     
     
         64 . The method according to  claim 62  or  claim 63 , wherein the non-complexed  botulinum  toxin A is eluted from the cation exchange column at a pH of 6 to 7. 
     
     
         65 . The method according to  claim 1  or  claim 43 , wherein the hydrophobic interaction column is a Phenyl Sepharose® HP hydrophobic interaction column; the anion exchange column is a Q XL Sepharose® anion exchange column; and the cation exchange column is a Source S cation exchange column; wherein the hydrophobic interaction column is eluted with a descending gradient of ammonium sulfate; and wherein the anion and cation exchange columns are eluted with an ascending gradient of sodium chloride. 
     
     
         66 . The method according to  claim 1 , wherein the purified non-complexed  botulinum  toxin A is at least 95% pure and/or has an activity of at least 200 LD 50  units/ng. 
     
     
         67 . The method according to any one of  claims 1 ,  11  or  66 , wherein said method produces a yield of at least about 1 mg to at least about 3 mg of purified non-complexed  botulinum  toxin A per liter (L) of fermentation culture. 
     
     
         68 . The method according to  claim 67 , wherein the method produces a yield of about 1 to about 2 mg per liter (L) of fermentation culture. 
     
     
         69 . The method according to  claim 67 , wherein the method produces a yield of at least about 3 mg of purified non-complexed  botulinum  toxin A per liter (L) of fermentation culture. 
     
     
         70 . The method according to  claim 67 , wherein the method produces a yield of at least about 2 mg of purified non-complexed  botulinum  toxin A per liter (L) of fermentation culture. 
     
     
         71 . The method according to  claim 67 , wherein the method produces a yield of at least about 1 mg of purified non-complexed  botulinum  toxin A per liter (L) of fermentation culture.