Large-scale production of human serum butyrylcholinesterase as a bioscavenger

Abstract

Disclosed herein are methods for the large-scale preparation of human butyrylcholinesterase (HuBChE) preparations from Cohn Fraction IV-4. As disclosed, the methods provide HuBChE preparations that are about 99% or more pure with recovery yields of about 60%. Also disclosed are the pharmacokinetics, safety and toxicity, stability and efficacy of the HuBChE preparations.

1 . A method for obtaining an amount of a human butyrylcholinesterase preparation which comprises subjecting an amount of Cohn Fraction IV-4 paste of about 2 or more kilograms to affinity chromatography followed by anion exchange chromatography.  
   
   
       2 . The method of  claim 1 , wherein the amount of Cohn Fraction IV-4 paste is about 10 to about 500 kilograms.  
   
   
       3 . The method of  claim 1 , wherein the amount of Cohn Fraction IV-4 paste is about 80 or more kilograms.  
   
   
       4 . The method of  claim 1 , which further comprises diluting the Cohn Fraction IV-4 paste by about ten-fold with water to obtain a suspension and then adjusting the suspension to a pH of about 4.5 to about 5.5.  
   
   
       5 . The method of  claim 4 , wherein the pH is about 4.9.  
   
   
       6 . The method of  claim 4 , which further comprises centrifuging the suspension in a continuous flow centrifuge at an rpm of about 7400 to about 7900 at a flow rate of about 2 to about 6 kilograms per minute to obtain a supernatant.  
   
   
       7 . The method of  claim 6 , wherein the flow rate is about 4 kilograms per minute.  
   
   
       8 . The method of  claim 6 , which further comprises adjusting the supernatant to a pH of about 7.0 to about 9.0.  
   
   
       9 . The method of  claim 8 , wherein the pH is about 8.0.  
   
   
       10 . The method of  claim 6 , which further comprises filtering the supernatant with a 0.65 μm filter cartridge.  
   
   
       11 . The method of  claim 1 , wherein the affinity chromatography and the anion exchange chromatography are performed once.  
   
   
       12 . The method of  claim 1 , wherein the affinity chromatography is conducted using a procainamide column.  
   
   
       13 . The method of  claim 1 , wherein the anion exchange chromatography is conducted using a DEAE sepharose fast flow column.  
   
   
       14 . The method of  claim 1 , wherein the amount of the human butyrylcholinesterase preparation obtained is about 60% w/w of that present in the Cohn Fraction IV-4 paste.  
   
   
       15 . The method of  claim 1 , wherein the human butyrylcholinesterase preparation is about 99% or more pure.  
   
   
       16 . The method of  claim 1 , wherein the human butyrylcholinesterase preparation is storage stable for more than two years at about −20° C. to about 45° C.  
   
   
       17 . The method of  claim 1 , wherein the pharmacokinetic parameters of purified enzyme are substantially the same after storage for about two years or more at about −20° C.  
   
   
       18 . The method of  claim 1 , wherein the human butyrylcholinesterase preparation is non-toxic.  
   
   
       19 . A human butyrylcholinesterase preparation made according to the method of  claim 1 .  
   
   
       20 . The human butyrylcholinesterase preparation of  claim 19 , packaged as a single dose in an autoinjector.  
   
   
       21 . A method of treating, preventing, or inhibiting toxicity of an organophosphorus compound in a subject which comprises administering to the subject the human butyrylcholinesterase preparation of  claim 19 .  
   
   
       22 . A pharmaceutical preparation comprising the human butyrylcholinesterase preparation of  claim 19  and a pharmaceutically acceptable carrier.