Animal product free system and process for purifying a botulinum toxin

Abstract

Chromatographic processes and systems for purifying a botulinum toxin from an APF fermentation medium.

1 - 6 . (canceled) 
     
     
         7 . A method for purifying and obtaining approximately 150 kDa  Clostridium botulinum  toxin serotype A (BoNT/A) in a sample comprising BoNT/A complex and at least one impurity protein, said method comprising:
 (a) contacting the sample with a first chromatography column to afford a first purified sample;   (b) contacting the first purified sample with a second chromatography column to afford a second purified sample;   (c) contacting the second purified sample with an aqueous solution having a pH between about 7.0 and about 8.0 on a third chromatography column to afford a third purified sample comprising dissociated approximately 150 kDa BoNT/A; and   (d) recovering the dissociated approximately 150 kDa BoNT/A.   
     
     
         8 . The method of  claim 7 , wherein the sample comprises a clarified culture. 
     
     
         9 . The method of  claim 7 , wherein the sample comprises a clarified culture extract. 
     
     
         10 . The method of  claim 8 , wherein the clarified culture has been prepared without an acid precipitation step. 
     
     
         11 . The method of  claim 7 , wherein the first chromatography column is selected from an ion exchange column, a hydrophobic interaction column, a gel filtration column, or a mixed mode column. 
     
     
         12 . The method of  claim 11 , wherein the first chromatography column is a hydrophobic interaction column. 
     
     
         13 . The method of  claim 7 , wherein the second chromatography column is an ion exchange column. 
     
     
         14 . The method of  claim 13 , wherein the ion exchange column is a cation exchange column. 
     
     
         15 . The method of  claim 7 , wherein the third chromatography column is an ion exchange column. 
     
     
         16 . The method of  claim 15 , wherein the ion exchange column is an anion exchange column. 
     
     
         17 . The method of  claim 7 , wherein step (a) further comprises contacting the sample with a first buffer on the first chromatography column. 
     
     
         18 . The method of  claim 17 , wherein the first buffer is a phosphate buffer. 
     
     
         19 . The method of  claim 17 , wherein the first buffer has a pH of from about 5.0 to about 6.0. 
     
     
         20 . The method of  claim 7 , wherein step (b) further comprises contacting the first purified sample with a second buffer on the second chromatography column. 
     
     
         21 . The method of  claim 20 , wherein the first buffer further comprises sodium chloride. 
     
     
         22 . The method of  claim 20 , wherein the first buffer has a pH of about 4.0.