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 - 14 . (canceled) 
     
     
         15 . A formulated  botulinum  toxin type A product that is free of bovine DNase, RNase, and casein, comprising:
 (i) a purified, approximately 150 kDa  botulinum  toxin type A molecule that is essentially free of animal products; and   (ii) at least one pharmaceutically acceptable excipient that is essentially free of animal products;   wherein the purified, approximately 150 kDa  botulinum  toxin type A molecule is obtained from a process comprising the steps of:
 (a) preparing a  botulinum  toxin fermentation culture comprising  botulinum  toxin type A, wherein the fermentation culture is essentially animal product free (APF); 
 (b) contacting a hydrophobic interaction chromatography (HIC) column resin with the  botulinum  toxin fermentation culture; 
 (c) eluting the  botulinum  toxin type A from the HIC column resin to provide a first eluent; 
 (d) conditioning the first eluent with a buffer having a pH of between about 7-8, whereby dissociated eluted  botulinum  toxin type A is formed comprising (1) at least one approximately 150 kDa  botulinum  toxin type A molecule and (2) at least one non toxin complex protein; 
 (e) contacting an anion exchange column resin with the first eluant; 
 (f) eluting the  botulinum  toxin type A from the anion exchange column resin to provide a second eluant; 
 (g) contacting a cation exchange column resin with the second eluant; and 
 (h) eluting the  botulinum  toxin type A from the cation exchange column to provide purified approximately 150 kDa  botulinum  toxin type A molecule. 
   
     
     
         16 . The formulated  botulinum  toxin type A product of  claim 15 , wherein the purified approximately 150 kDa  botulinum  toxin type A molecule has a potency that meets or exceeds the potency of a purified  botulinum  toxin product obtained from a Schantz or a modified Schantz process. 
     
     
         17 . The formulated  botulinum  toxin type A product of  claim 15 , wherein the purified approximately 150 kDa  botulinum  toxin type A molecule has a potency that exceeds the potency of a purified  botulinum  toxin product obtained from a Schantz or a modified Schantz process. 
     
     
         18 . The formulated  botulinum  toxin type A product of  claim 15 , wherein the purified approximately 150 kDa  botulinum  toxin type A molecule has a specific potency of 1-2×10 8  LD 50  U/mg or greater. 
     
     
         19 . The formulated  botulinum  toxin type A product of  claim 15 , wherein more than 99% of non- botulinum  toxin proteins and impurities are removed. 
     
     
         20 . The formulated  botulinum  toxin type A product of  claim 15 , wherein the formulated  botulinum  toxin type A product is essentially free of animal product (APF). 
     
     
         21 . The formulated  botulinum  toxin type A product of  claim 15 , wherein the formulated  botulinum  toxin type A product is free of animal product (APF). 
     
     
         22 . The formulated  botulinum  toxin type A product of  claim 15 , wherein there is no more than 6 ng/ml residual DNA from  Clostridium botulinum  bacterium. 
     
     
         23 . The formulated  botulinum  toxin type A product of  claim 15 , wherein the fermentation culture is prepared with a fermentation medium comprising about 10-100 g/L hydrolyzed soy protein. 
     
     
         24 . The formulated  botulinum  toxin type A product of  claim 15 , wherein the fermentation culture is prepared with a fermentation medium comprising about 6 wt % of hydrolyzed soy protein. 
     
     
         25 . The formulated  botulinum  toxin type A product of  claim 15 , wherein the fermentation culture is prepared with a fermentation medium comprising about 1 wt % of yeast extract. 
     
     
         26 . The formulated  botulinum  toxin type A product of  claim 15 , wherein the fermentation culture is prepared with a fermentation medium comprising about 1 wt % of glucose. 
     
     
         27 . The formulated  botulinum  toxin type A product of  claim 15 , wherein the fermentation culture is prepared with a fermentation medium comprising about 6 wt % of hydrolyzed soy protein, about 1 wt % of yeast extract, and about 1 wt % of glucose. 
     
     
         28 . The formulated  botulinum  toxin type A product of  claim 15 , wherein a yield of greater than about 50 mg  botulinum  toxin complex for each 10 liters of the  botulinum  toxin fermentation culture is obtained. 
     
     
         29 . The formulated  botulinum  toxin type A product of  claim 15 , wherein a potency of about 38×10 5  LD 50  units/mL of  botulinum  toxin fermentation culture is obtained. 
     
     
         30 . A method for obtaining a biologically active  botulinum  toxin type A, comprising the steps of:
 (a) providing a fermentation medium which is free of an animal product;   (b) culturing a  Clostridium botulinum  type A bacterium in the fermentation medium under conditions which permit production of a  botulinum  toxin type A, and;   (c) recovering a biologically active  botulinum  toxin type A from the fermentation medium,   wherein the fermentation medium comprises a protein product obtained from yeast or from a vegetable and wherein the vegetable is selected from the group consisting of soy, malt and corn.   
     
     
         31 . A method for making an animal product free pharmaceutical composition in which the active ingredient is a  botulinum  toxin type A, the method comprising the steps of:
 (a) obtaining a biologically active  botulinum  toxin type A by:
 i. providing a fermentation medium which is free of an animal product; 
 ii. culturing a  Clostridium botulinum  type A bacterium in the fermentation medium under conditions which permit production of a  botulinum  toxin type A, and; 
 iii. recovering a biologically active  botulinum  toxin type A from the fermentation medium; 
   (b) formulating the  botulinum  toxin type A with a suitable excipient, thereby making an animal product free pharmaceutical composition in which the active ingredient is a  botulinum  toxin type A,   wherein the fermentation medium comprises a protein product obtained from yeast or from a vegetable and wherein the vegetable is selected from the group consisting of soy, malt and corn.   
     
     
         32 . An animal protein free (“APF”) process for purifying a biologically active  botulinum  toxin, the process comprising the steps of:
 (a) obtaining a sample of a  botulinum  toxin fermentation culture, wherein the  botulinum  toxin fermentation culture results from a substantially APF process, 
 (b) contacting a hydrophobic interaction chromatography column resin with the culture sample so as to permit capture of a  botulinum  toxin by the hydrophobic interaction chromatography column; 
 (c) washing impurities off the hydrophobic interaction chromatography column; 
 (d) eluting the  botulinum  toxin from the hydrophobic interaction column; 
 (e) loading an ion exchange column chromatography column resin with the eluent from the hydrophobic interaction chromatography column; 
 (f) washing impurities off the ion exchange chromatography column, and; 
 (g) eluting the  botulinum  toxin from the ion exchange column, thereby obtaining a purified biologically active  botulinum  toxin through a process for purifying a  botulinum  toxin which is a substantially APF purification process.