Novel prongf mutants and uses thereof in the production of beta-ngf

Abstract

The present invention relates to a proNGF mutant and to uses thereof, in particular the use of a proNGF mutant for producing human beta-NGF. The present invention discloses a method of preparing a biologically active human beta-NGF from an inactive insoluble proNGF mutant. A proNGF mutant of the invention is substituted by amino acid but not Arg or Lys at the native protease cleavage site R1SK3R4 at least at positions R1 and K3 corresponding to positions 101 and 103 of the human wildtype proNGF sequence.

1 . A biologically active human beta-NGF produced by:
 a. providing a proNGF mutant wherein a protease cleavage site R1SK3R4 of the proNGF mutant is substituted at least at positions R1 and K3 corresponding to positions 101 and 103 of the human wild type proNGF sequence (SEQ ID NO: 1) by any amino acid selected from the group consisting of a non-basic amino acid and Histidine; and   b. cleaving the pro-sequence of the proNGF mutant.   
     
     
         2 . The biologically active human beta-NGF of  claim 1  produced by:
 a. providing a proNGF mutant wherein a protease cleavage site R1SK3R4 of the proNGF mutant is substituted at least at positions R1 and K3 corresponding to positions 101 and 103 of the human wild type proNGF sequence (SEQ ID NO: 1) by any amino acid selected from the group consisting of a non-basic amino acid and Histidine, and wherein the amino acid at position R4 corresponding to position 104 of human wild type proNGF sequence (SEQ ID NO: 1) is selected from the group consisting of Arginine and Lysine; and 
 b. cleaving the pro-sequence of the proNGF mutant by a serine protease. 
 
     
     
         3 . A biologically active human beta-NGF produced by:
 a. providing a proNGF mutant wherein a protease cleavage site R1SK3R1 of the proNGF mutant is substituted at least at positions R1 and K3 corresponding to positions 101 and 103 of the human wild type proNGF sequence (SEQ ID NO: 1) by any amino acid selected from the group consisting of a non-basic amino acid and Histidine; and   b. cleaving the pro-sequence of the proNGF mutant by a serine protease.   
     
     
         4 . A biologically active human beta-NGF produced by:
 a. providing a proNGF mutant wherein the protease cleavage site R4SK3R4 of the proNGF mutant is substituted at least at positions R1 and K3 corresponding to positions 101 and 103 of the human wild type proNGF sequence (SEQ ID NO: 1) by any amino acid selected from the group consisting of a non-basic amino acid and Histidine, and wherein the amino acid at position R4 corresponding to position 104 of human wild type proNGF sequence (SEQ ID NO: 1) is selected from the group consisting of Arginine and Lysine; and   b. cleaving the pro-sequence of the proNGF mutant by a serine protease in order to obtain active human beta-NGF, wherein the ratio of serine protease to proNGF mutant is 1:5,000-1:20,000 (w/w), 1:10,000 (w/w), or 1:100,000 (w/w).   
     
     
         5 . The biologically active human beta-NGF of  claim 1 , further comprising:
 a. dissolving the proNGF mutant by solubilisation of inclusion bodies in a denaturating solution;   b. transferring the proNGF mutant into a refolding solution where the denatured proNGF assumes a biologically active conformation; and   c. purifying the refolded proNGF mutant.   
     
     
         6 . The biologically active human beta-NGF of  claim 5 , wherein the denaturing solution comprises a solution containing (i) a chaotropic substance, (ii) a chelator, (iii) a buffer, and (iv) a reducing agent. 
     
     
         7 . The biologically active human beta-NGF of  claim 5 , wherein the refolding is carried out as a pulse renaturation. 
     
     
         8 . The biologically active human beta-NGF of  claim 5 , wherein the proNGF mutant is purified via mixed mode chromatography. 
     
     
         9 . The biologically active human beta-NGF of  claim 3 , further comprising:
 a. dissolving the proNGF mutant wherein the protease cleavage site R1SK3R4 is substituted at least at positions R1 and K3 corresponding to positions 101 and 103 of the human wild type proNGF sequence (SEQ ID NO: 1) by an amino acid selected from the group consisting of a non-basic amino acid and Histidine by solubilization of inclusion bodies in a denaturing solution;   b. transferring the proNGF mutant into a refolding solution where the denatured proNGF assumes a biologically active conformation;   c. purifying the refolded proNGF mutant; and   d. cleaving the pro-sequence of the proNGF mutant to obtain the active beta-NGF.   
     
     
         10 . The biologically active human beta-NGF of  claim 6 , wherein the denaturing solution used in production of the biological active human beta-NGF comprises:
 i. 1-8 M Guanidinium-HCl,   ii. 0.01-1 M Tris,   iii. 1-50 mM EDTA,   iv. 1-100 mM selected from Gluthione (GSH) or Cysteine,   v. pH 7.0-10.0.   
     
     
         11 . The biologically active human beta-NGF of  claim 10 , wherein 1-8 M Guanidinium-HCl further comprises 4-6 M Guanidinium-HCl. 
     
     
         12 . The biologically active human beta-NGF  claim 6 , wherein the denaturing solution used in production of the biological active human beta-NGF comprises:
 i. 4 M Guanidinium-HCL   ii. 0.1 M Tris,   iii. 10 mM EDTA,   iv. 5 mM GSH or Cysteine,   v. pH 8.0.   
     
     
         13 . The biologically active human beta-NGF of  claim 5 , wherein the refolding solution used in production of the biologically active human beta-NGF comprises:
 i. 0.5-1.0 M of a chaperone,   ii. 1-10 mM of a metal chelator,   iii. 0.1-10 mM of a redox shuffling system,   iv. pH 8.0-pH 11.0.   
     
     
         14 . The biologically active human beta-NGF of  claim 13 , wherein the refolding solution used in production of the biologically active human beta-NGF further comprises:
 i. 0.75 M Arginine,   ii. 5 mM EDTA   iii. 1 mM L-Cystine and 5 mM L-Cysteine, or 1 mM GSSG (oxidized glutathione) and 5 mM GSH (reduced glutathione),   iv. pH 9.5.   
     
     
         15 . The biologically active human beta-NGF of  claim 8 , wherein the proNGF mutant, used to produce the biologically active human beta-NGF, is purified using a mixed mode material chromatography column with a synthetic affinity ligand. 
     
     
         16 . The biologically active human beta-NGF of  claim 15 , wherein the chromatography column, used to purify the proNGF mutant used in production of the biologically active human beta-NGF, is a column with a synthetic affinity ligand chosen from the group consisting of 4-mercapto-ethyl-pyridine (MEP), Hexylamino (HEA), Phenylpropylamino (PPA), 2-Mercapto-5-benzamidazole sulfo acid (MB I), Capto MMC (GEHC), N-benzyl-N-methyl ethanolamine (GEHC)), CHT hydroxyapatide and CHT fluoroapatide. 
     
     
         17 . The biologically active human beta-NGF of  claim 16 , wherein chromatography column, used to purify the proNGF mutant used in production of the biologically active human beta-NGF, is a column with a synthetic affinity ligand that is 4-mercapto-ethyl-pyridine (MEP). 
     
     
         18 . A method of using human betaNGF, produced using a proNGF mutant, to administer medical treatments to patients, including:
 a. substituting the protease cleavage site R1SK3R4 of the proNGF mutant at R1 and K3, corresponding to positions 101 and 103 of human wild type proNGF sequence (SEQ ID NO: 1), by any amino acid selected from the group consisting of a non-basic amino acid and Histidine; and   b. selecting the amino acid at position R4 of the proNGF mutant, corresponding to position 104 of human wild type proNGF sequence (SEQ ID NO: 1), from the group consisting of Arginine or Lysine.   
     
     
         19 . The method according to  claim 18  further including:
 a. producing a pharmaceutically active composition, of betaNGF obtained from proNGF; 
 b. providing a beta-NGF obtained from a proNGF mutant including:
 i. substituting the protease cleavage site R1SK3R4 of the proNGF mutant at R1 and K3, corresponding to positions 101 and 103 of human wild type proNGF sequence (SEQ ID NO: 1), by any amino acid selected from the group consisting of a non-basic amino acid and Histidine; and 
 ii. selecting the amino acid at position R4 of the proNGF mutant, corresponding to position 104 of human wild type proNGF sequence (SEQ ID NO: 1), from the group consisting of Arginine or Lysine; and 
 
 c. providing a pharmaceutically acceptable carrier. 
 
     
     
         20 . Biologically active human beta-NGF, characterized by being substantially free from impurities, said impurities consisting of at least cleavage by-products typically associated with beta-NGF obtained from wild type proNGF. 
     
     
         21 . The biologically active human beta-NGF of  claim 20 , wherein said impurities, of which the biological active human beta-NGF is substantially free from, consist of by-products typically associated with beta-NGF obtained from wild type proNGF, and trypsin. 
     
     
         22 . The biologically active human beta-NGF of  claim 20 , having a purity of at least 97%. 
     
     
         23 . A method of using biologically active human beta-NGF, characterized by being substantially free from impurities, said impurities consisting of at least cleavage by-products typically associated with beta-NGF obtained from wild type proNGF, to administer medical treatments to patients. 
     
     
         24 . A method of producing a pharmaceutically active composition of biologically active human beta-NGF, characterized by being substantially free from impurities, said impurities consisting of at least cleavage by-products, typically associated with beta-NGF, obtained from wild type proNGF, produced by providing a pharmaceutically acceptable carrier.