Botulinum antitoxin compositions and methods

Abstract

This invention provides botulinum antitoxin compositions and methods of production, and methods of treating animals and humans prophylactically and also those suspected of having contacted botulism toxin. The botulinum antitoxin is prepared by inoculating an animal with a monovalent botulinum toxoid and toxin. The animal's plasma is collected and purified at a high pH by affinity chromatography. The resulting monovalent immunoglobulins are de-speciated by digestion with pepsin. Monovalent antitoxins for all seven botulinum serotypes are then combined to produce a high titered heptavalent botulinum antitoxin composition.

1 . A method for the manufacture of a  botulinum  antitoxin composition comprising: 
 injecting an animal with a monovalent  botulinum  toxoid and toxin to produce immunoglobulins and collecting plasma containing the immunoglobulins from the animal;    purifying the immunoglobulins from the plasma by affinity chromatography; and    digesting the purified immunoglobulins by a proteolytic enzyme to obtain a monovalent  botulinum  antitoxin composition.    
     
     
         2 . The method of  claim 1 , wherein the animal is a horse.  
     
     
         3 . The method of  claim 1 , further comprising combining a plurality of monovalent  botulinum  antitoxins for different  botulinum  toxins to create a polyvalent  botulinum  antitoxin composition.  
     
     
         4 . The method of  claim 3 , wherein the polyvalent  botulinum  antitoxin composition comprises antitoxin for seven different  botulinum  toxins.  
     
     
         5 . The method of  claim 4 , wherein the antitoxins for serotypes A, B, C, E, and F have a potency >4,000 International Units and the antitoxins for serotypes D and G have a potency >500 International Units.  
     
     
         6 . The method of  claim 1 , wherein the affinity chromatography uses immobilized Protein G.  
     
     
         7 . The method of  claim 3  further comprising supplementing the polyvalent  botulinum  antitoxin with  botulinum  monoclonal antibodies.  
     
     
         8 . The method of  claim 7 , wherein the monoclonal antibodies are produced from mouse myeloma cells and equine lymphocyte hybridomas.  
     
     
         9 . The method of  claim 7 , wherein the monoclonal antibodies are directed against  botulinum  neurotoxins selected from the group consisting of neurotoxins F and G.  
     
     
         10 . The method of  claim 1 , wherein the animal is injected intradermally with toxoid.  
     
     
         11 . The method of  claim 1  further comprising injecting the animal with  botulinum  toxin after the toxoid injections and before collecting the plasma.  
     
     
         12 . The method of  claim 1 , wherein the injected toxoid further comprises an adjuvant.  
     
     
         13 . The method of  claim 1 , wherein the animal is injected with a first injection of toxoid and a second injection of toxoid.  
     
     
         14 . The method of  claim 13 , wherein the first injection comprises about 2 mg of toxoid.  
     
     
         15 . The method of  claim 13 , wherein the first injection is injected at multiple sites at about 0.1 mL per site.  
     
     
         16 . The method of  claim 13 , wherein the first injection further comprises Complete Freund's Adjuvant.  
     
     
         17 . The method of  claim 13 , wherein the second injection is given about 14 days after the first injection.  
     
     
         18 . The method of  claim 13 , wherein the second injection comprises about 0.5 mg of toxoid.  
     
     
         19 . The method of  claim 13 , wherein the second injection further comprises Incomplete Freund's Adjuvant.  
     
     
         20 . The method of  claim 13 , wherein the second injection is injected at multiple sites at approximately 0.1 mL per site.  
     
     
         21 . The method of  claim 13 , wherein a priming dose of toxin is injected after the second injection of toxoid.  
     
     
         22 . The method of  claim 1 , wherein the animal is injected with purified toxin about 7 to 10 days before the plasma is collected.  
     
     
         23 . The method of  claim 1 , wherein the animal is injected with purified toxin conjugated to Keyhole limpet hemocyanin about 7 to 10 days before the plasma is collected.  
     
     
         24 . The method of  claim 1 , further comprising clarifying the plasma through a filter after the plasma is collected.  
     
     
         25 . The method of  claim 24 , wherein the filter comprises pore sizes selected from the group consisting of 2.0 μ, 1.2 μ, 0.5 μ, and 0.22 μ.  
     
     
         26 . The method of  claim 1 , wherein the affinity chromatography is performed at a pH between about pH 10-12.  
     
     
         27 . The method of  claim 1 , wherein the affinity chromatography is performed at about pH 11.  
     
     
         28 . The method of  claim 1 , wherein pepsin is used to digest the purified immunoglobulins.  
     
     
         29 . The method of  claim 1 , wherein the digesting is performed at a pH between about pH 2.5-6.0.  
     
     
         30 . The method of  claim 29 , wherein the pH is about pH 4.5.  
     
     
         31 . The method of  claim 1 , wherein the immunoglobulins are digested at a temperature of about 20-70° C.  
     
     
         32 . The method of  claim 31 , wherein the temperature is about 58° C.  
     
     
         33 . The method of  claim 1 , wherein the digested immunoglobulins are concentrated to about 90-100 mg/mL protein.  
     
     
         34 . The method of  claim 1 , wherein the digested immunoglobulins are purified on an anion exchange column.  
     
     
         35 . The method of  claim 1 , further comprising lyophilizing the antitoxin composition.  
     
     
         36 . A  botulinum  antitoxin composition prepared by the method of  claim 1 .  
     
     
         37 . The  botulinum  antitoxin composition of  claim 36 , having a pH in the range of about 6-8.  
     
     
         38 . The  botulinum  antitoxin composition of  claim 36 , wherein the composition has a purity of at least about 95%.  
     
     
         39 . The  botulinum  antitoxin composition of  claim 36 , wherein the composition has a protein concentration in the range of about 30-70 mg/ml.  
     
     
         40 . The  botulinum  antitoxin composition of  claim 36 , wherein the composition comprises at least about 60% F(ab′) 2  and about 40% or less of Fab′ or Fab.  
     
     
         41 . A method for the manufacture of a heptavalent  botulinum  antitoxin composition comprising: 
 injecting a plurality of horses with monovalent  botulinum  toxoid mixed with an adjuvant,    injecting the horses with toxin after the injections of toxoid;    collecting from the horses plasma containing immunoglobulins and purifying the immunoglobulins from the plasma by affinity chromatography using immobilized Protein G, wherein the chromatography is performed at about pH 11;    digesting the purified immunoglobulins with pepsin at a temperature of about 58° C. and a pH of about 4.5;    filtering the digested immunoglobulins to obtain a monovalent  botulinum  antitoxin;    combining the monovalent  botulinum  antitoxins from the plurality of horses to create a polyvalent composition;    supplementing the polyvalent composition with monoclonal antibodies directed against  botulinum  toxins F and G to produce a heptavalent composition; and    lyophilizing the heptavalent composition.    
     
     
         42 . A method of treating an animal in need of  botulinum  antitoxin by administering an effective amount of the antitoxin produced by the method of  claim 1 .  
     
     
         43 . The method of  claim 42  wherein the antitoxin is administered intravenously.  
     
     
         44 . A method of preventing  Clostridium botulinum  poisoning comprising administering an effective amount of the antitoxin produced by the method of  claim 1 .  
     
     
         45 . A  botulinum  antitoxin composition comprising antitoxin for  botulinum  toxins A, B, C, D, E, F, and G.  
     
     
         46 . The composition of  claim 45 , wherein the antitoxins for  botulinum  toxins A, B, C, E, and F have a potency >4,000 I.U. and the antitoxins for toxins D and G have a potency >500 I.U.  
     
     
         47 . The composition of  claim 45 , wherein the composition comprises about 95% of at least one member selected from the group consisting of F(ab′) 2 , Fab, and Fab′.  
     
     
         48 . The composition of  claim 45 , wherein the composition comprises at least about 60% F(ab′) 2  and about 40% or less of Fab′ or Fab.