Chromatographic method and system for purifying a botulinum toxin

Abstract

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

1 . A method for obtaining a biologically active botulinum toxin, comprising the steps of:
 (a) providing a fermentation medium that is substantially free of an animal derived product;   (b) culturing a  Clostridium botulinum  bacterium in the fermentation medium under conditions which permit production of a botulinum toxin, and;   (c) recovering a biologically active botulinum toxin from the fermentation medium.   
     
     
         2 . The method of  claim 1 , wherein in the step of providing a fermentation medium, the medium comprises a protein product derived from a vegetable. 
     
     
         3 . The method of  claim 2 , wherein the vegetable is a soybean. 
     
     
         4 . The method of  claim 1 , wherein in the step of culturing, the culturing is performed until cell density of the culture decreases due to cell lysis. 
     
     
         5 . The method of  claim 1 , wherein in the step of culturing, the culturing is performed until at least 48 hours after initial drop in cell density due to cell lysis. 
     
     
         6 . A method for production of a botulinum toxin, the method comprising steps of:
 (a) providing a first medium that is substantially free of an animal derived product and that comprises a protein product derived from a vegetable; (b) culturing a  Clostridium botulinum  bacterium the first medium under conditions that permit growth of the  Clostridium botulinum;      (c) providing a second medium that is substantially free of an animal derived product and that comprises a protein product derived from a vegetable;   (d) inoculating the second medium with the first medium;   (e) culturing a  Clostridium botulinum  bacterium in the second medium under conditions that allow production of a botulinum toxin, and;   (f) recovering the botulinum toxin.   
     
     
         7 . The method of  claim 6 , wherein in the step of providing a first medium, the first medium comprises a protein product derived from a soybean, and wherein in the step of inoculating the second medium, the second medium comprises the protein product derived from a soybean. 
     
     
         8 . The method of  claim 6 , wherein in the step of providing a first medium, the first medium comprises a hydrolyzed soy,
 and wherein in the step of inoculating a second medium, the fermentation medium comprises hydrolyzed soy.   
     
     
         9 . The method of  claim 6 , wherein in the step of culturing a  Clostridium botulinum  in a first medium, the conditions comprise a temperature of about 34 degrees Celsius, and further comprise no decrease in cell density during culturing, wherein in the step of inoculating a second medium with a first medium, 2 to 4 percent of the first medium is used to inoculate the second medium, and wherein in the step of culturing the bacterium in the second medium, the conditions that allow growth comprise a temperature of approximately 34 degrees Celsius and further comprise culturing until cell density of the culture decreases due to cell lysis. 
     
     
         10 . A composition comprising a  Clostridium botulinum  and a culture medium for producing a botulinum toxin wherein the medium is substantially free of an animal derived product and comprises a protein product derived from a vegetable. 
     
     
         11 . The composition of  claim 10 , wherein the vegetable is a soybean. 
     
     
         12 . The composition of  claim 10 , wherein the composition comprises a hydrolyzed soy. 
     
     
         13 . A method for making a substantially animal product free pharmaceutical composition in which the active ingredient is a botulinum toxin, the method comprising the steps of:
 (a) obtaining a biologically active botulinum toxin by;
 (i) providing a fermentation medium that is substantially free of an animal derived product; 
 (ii) culturing a  Clostridium botulinum  in the fermentation medium under conditions which permit production of a botulinum toxin, and; 
 (iii) recovering a biologically active botulinum toxin from the fermentation medium; 
   (b) formulating the botulinum toxin with a suitable excipient, thereby making a substantially animal product free pharmaceutical composition in which the active ingredient is a botulinum toxin.