Composition comprising recombinant clostridium neurotoxin

Abstract

The invention provides methods for producing soluble di-chain BoNT/A protein.

1 . A composition comprising an active chain BoNT/A protein obtainable by a method comprising:
 (a) providing an isolated soluble single-chain BoNT/A protein;   (b) contacting said BoNT/A protein with endoproteinase Lys-C (Lys-C) in solution to produce a solution comprising the active di-chain BoNT/A protein and Lys-C; and   (c) separating the active di-chain BoNT/A protein from the Lys-C by contacting the solution containing the active di-chain BoNT/A protein and Lys-C with a hydrophobic surface, wherein the active di-chain BoNT/A protein preferentially binds to the hydrophobic surface;   
       and wherein said composition comprises (i) less than 2% single-chain BoNT/A; and (ii) Lys-C, wherein the Lys-C is present at a concentration of less than 400 pg Lys-C per 100 ng BoNT/A protein. 
     
     
         2 - 12 . (canceled) 
     
     
         13 . The composition according to  claim 1 , wherein said composition comprises less than 1% single chain BoNT/A. 
     
     
         14 . The composition according to  claim 1 , wherein said composition contains endoproteinase Lys-C (Lys C) at a concentration of less than 300 pg Lys-C per 100 ng BoNT/A protein. 
     
     
         15 . A liquid pharmaceutical composition comprising:
 (a) an active di-chain BoNT/A protein obtainable by a method comprising:
 (i) providing an isolated soluble single-chain BoNT/A protein; 
 (ii) contacting said BoNT/A protein with endoproteinase Lys-C (Lys-C) in solution to produce a solution comprising the active di-chain BoNT/A protein and Lys-C; and 
 (iii) separating the active di-chain BoNT/A protein from the Lys-C by contacting the solution containing the active di-chain BoNT/A protein and Lys-C with a hydrophobic surface, wherein the active di-chain BoNT/A protein preferentially binds to the hydrophobic surface; 
   (b) a non-ionic surfactant; and   (c) water;   
       and wherein said liquid pharmaceutical composition:
 (i) does not comprise a protein stabilising agent; 
 (ii) comprises less than 2% single-chain BoNT/A; and 
 (iii) comprises Lys-C, wherein the Lys-C is present at a concentration of less than 400 pg Lys-C per 100 ng BoNT/A protein. 
 
     
     
         16 . The liquid pharmaceutical composition according to  claim 15 , wherein said liquid pharmaceutical composition contains endoproteinase Lys-C (Lys-C) at a concentration of less than 300 pg Lys-C per 100 ng BoNT/A protein. 
     
     
         17 . The liquid pharmaceutical composition according to  claim 15 , wherein said liquid pharmaceutical composition further comprises:
 sodium chloride;   a buffer to maintain pH between 5.5 and 7.5; and   a disaccharide; and   
       wherein the water is sterile water. 
     
     
         18 - 20 . (canceled) 
     
     
         21 . The composition according to  claim 1 , wherein said composition contains endoproteinase Lys-C (Lys-C) at a concentration of less than 200 pg Lys-C per 100 ng BoNT/A protein. 
     
     
         22 . The composition according to  claim 1 , wherein said composition contains endoproteinase Lys-C (Lys-C) at a concentration of less than 100 pg Lys-C per 100 ng BoNT/A protein. 
     
     
         23 . The composition according to  claim 1 , wherein said composition contains endoproteinase Lys-C (Lys-C) at a concentration of less than 50 pg Lys-C per 100 ng BoNT/A protein. 
     
     
         24 . The composition according to  claim 1 , wherein said composition contains endoproteinase Lys-C (Lys-C) at a concentration of less than 20 pg Lys-C per 100 ng BoNT/A protein. 
     
     
         25 . The composition according to  claim 1 , wherein said composition contains endoproteinase Lys-C (Lys-C) at a concentration of less than 10 pg Lys-C per 100 ng BoNT/A protein. 
     
     
         26 . The liquid pharmaceutical composition according to  claim 15 , wherein said liquid pharmaceutical composition contains endoproteinase Lys-C (Lys-C) at a concentration of less than 200 pg Lys-C per 100 ng BoNT/A protein. 
     
     
         27 . The liquid pharmaceutical composition according to  claim 15 , wherein said liquid pharmaceutical composition contains endoproteinase Lys-C (Lys-C) at a concentration of less than 100 pg Lys-C per 100 ng BoNT/A protein. 
     
     
         28 . The liquid pharmaceutical composition according to  claim 15 , wherein said liquid pharmaceutical composition contains endoproteinase Lys-C (Lys-C) at a concentration of less than 50 pg Lys-C per 100 ng BoNT/A protein. 
     
     
         29 . The liquid pharmaceutical composition according to  claim 15 , wherein said liquid pharmaceutical composition contains endoproteinase Lys-C (Lys-C) at a concentration of less than 20 pg Lys-C per 100 ng BoNT/A protein. 
     
     
         30 . The liquid pharmaceutical composition according to  claim 15 , wherein said liquid pharmaceutical composition contains endoproteinase Lys-C (Lys-C) at a concentration of less than 10 pg Lys-C per 100 ng BoNT/A protein. 
     
     
         31 . The liquid pharmaceutical composition according to  claim 15 , wherein said non-ionic surfactant, is a polysorbate or poloxamer. 
     
     
         32 . The liquid pharmaceutical composition according to  claim 15 , wherein said surfactant is present at a concentration of less than 1% v/v. 
     
     
         33 . The liquid pharmaceutical composition according to  claim 17 , wherein:
 (a) said disaccharide is selected from the group consisting of: sucrose, trehalose, mannitol and lactose; and/or   (b) the concentration of said disaccharide is from 5 to 50 mM.   
     
     
         34 . The liquid pharmaceutical composition according to  claim 17 , wherein:
 (a) said buffer is selected from group consisting of: succinate, disodium phosphate/citric acid, and an amino such as histidine; and/or   (b) the concentration of said buffer is from 1 to 50 mM, preferably from 5 to 20 mM.   
     
     
         35 . The composition according to  claim 1 , wherein the protease function of the active di-chain BoNT/A protein remains stable at 25° C. for at least 12 weeks. 
     
     
         36 . A method of treatment comprising administering a therapeutically effective amount of a composition according to  claim 1  to an individual in need thereof. 
     
     
         37 . A method of treating or preventing at least one of the following diseases and disorders: glabellar lines or hyperkinetic facial lines, headache, hemifacial spasm, hyperactivity of the bladder, hyperhidrosis, nasal labial lines, cervical dystonia, blepharospasm, and spasticity, said method comprising administering a therapeutically effective amount of the composition according to  claim 1  to an individual in need thereof. 
     
     
         38 . The liquid pharmaceutical composition according to  claim 15 , wherein the protease function of the active di-chain BoNT/A protein remains stable at 25° C. for at least 12 weeks. 
     
     
         39 . A method of treatment comprising administering a therapeutically effective amount of the liquid pharmaceutical composition according to  claim 15  to an individual in need thereof. 
     
     
         40 . A method of treating or preventing at least one of the following diseases and disorders: glabellar lines or hyperkinetic facial lines, headache, hemifacial spasm, hyperactivity of the bladder, hyperhidrosis, nasal labial lines, cervical dystonia, blepharospasm, and spasticity, said method comprising administering a therapeutically effective amount of the liquid pharmaceutical composition according to  claim 15  to an individual in need thereof.