Separation of vwf and vwf propeptide by chromatographic methods

Abstract

The present invention relates to a method for separating a mature von Willebrand Factor (mat-VWF) from von Willebrand Factor pro-peptide (VWF-PP) by incubating a composition comprising inducing dissociation of mat-VWF and VWF-PP by disruption of the non-covalently associated mat-VWF and VWF-PP, wherein said dissociation is induced by: (i) addition of at least one chelating agent, or (ii) increasing the pH to a pH of at least 7, and then collecting said mat-VWF to obtain a high purity, propeptide depleted mature VWF (mat-VWF).

1 .- 78 . (canceled) 
     
     
         79 . A pharmaceutical composition comprising a high purity mat-rVWF and a pharmaceutically acceptable buffer, wherein the high purity mat-rVWF is produced by a method comprising:
 a) loading a solution comprising mat-rVWF/rVWF-PP complex, mat-rVWF, and rVWF propeptide (rVWF-PP) onto a size exclusion column;   b) washing said size exclusion column with a buffer, thereby dissociating said mat-rVWF/rVWF-PP complex in said solution in step (a) into mat-rVWF and rVWF-PP, wherein said dissociation occurs by disruption of the non-covalently associated mat-rVWF and rVWF-PP, wherein said buffer comprises at least one chelating agent and exhibits a pH of at least 7; and   c) collecting said mat-rVWF to obtain a high purity, mat-rVWF, wherein said high purity, mat-rVWF composition comprises at least 95% mature rVWF and less than 5% rVWF-PP.   
     
     
         80 . The pharmaceutical composition of  claim 79 , wherein the composition comprises 50 mM Glycine, 10 mM Taurine, 5% (w/w) Sucrose, 5% (w/w) D-Mannitol, 0.1% Polysorbate 80, 2 mM CaCl 2 ), 150 mM NaCl, wherein said composition has a pH of about pH 7.4. 
     
     
         81 . The pharmaceutical composition of  claim 79 , wherein said high purity, mat-rVWF composition comprises at least 96% mat-rVWF and less than 4% rVWF-PP, at least 97% mat-rVWF and less than 3% rVWF-PP, at least 98% mat-rVWF and less than 2% rVWF-PP, at least 99% mat-rVWF and less than 1% rVWF-PP, or at least 99.5% mat-rVWF and less than 0.5% rVWF-PP, or 99.9% mat-rVWF and less than 0.1% rVWF-PP. 
     
     
         82 . The pharmaceutical composition of  claim 79 , wherein said solution of step (b) is selected from the group consisting of a cell culture medium, an antibody column flow-through solution, and a buffered solution. 
     
     
         83 . The pharmaceutical composition of  claim 79 , wherein said solution has been treated with furin prior to step (a) and/or wherein said solution is an antibody column flow-through solution. 
     
     
         84 . The pharmaceutical composition of  claim 79 , wherein said at least one chelating agent of step (b) is a divalent cation chelating agent. 
     
     
         85 . The pharmaceutical composition of  claim 84 , wherein said divalent cation chelating agent is selected from the group consisting of EDTA, EGTA, CDTA, and citrate 
     
     
         86 . The pharmaceutical composition of  claim 79 , wherein said pH is at least 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, or 9.0. 
     
     
         87 . The pharmaceutical of  claim 79 , wherein said pH is increased by the addition of basic amino acids, Tris, NaOH, Tricine, or ethanolamine. 
     
     
         88 . The pharmaceutical composition of  claim 79 , wherein said buffer comprises a buffering agent selected from the group consisting of glycine, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), TrisHCl (Tris(hydroxymethyl)-aminomethane), histidine, imidazole, acetate citrate, MES, and 2-(N-morpholino)ethanesulfonic acid. 
     
     
         89 . The pharmaceutical composition of  claim 79 , wherein said buffer further comprises one or more monovalent cations. 
     
     
         90 . The pharmaceutical composition of  claim 89 , wherein said one or more monovalent cations are selected from the group consisting of Na+, K+, Li+, and Cs+. 
     
     
         91 . The pharmaceutical composition of  claim 79 , wherein said buffer further comprises one or more monovalent, divalent and/or trivalent anion. 
     
     
         92 . The pharmaceutical composition of  claim 91 , wherein said one or more monovalent, divalent and/or trivalent anions are selected from the group consisting of Cl − , acetate − , SO 4   2− , Br − , and citrate 3− . 
     
     
         93 . The pharmaceutical composition of  claim 79 , wherein said solution comprising mat-rVWF/rVWF-PP complex, mat-rVWF, and rVWF-PP is derived from a capture step for rVWF or derived from a method comprising a FVIII immunoaffinity step and anion exchange chromatography step. 
     
     
         94 . The pharmaceutical composition of  claim 79 , wherein said buffer comprises a buffering agent(s) selected from the group consisting of (i) Na citrate, (ii) NaCl, and (iii) HEPES, Na citrate, and NaCl. 
     
     
         95 . The pharmaceutical composition of  claim 79 , wherein said method further comprises lyophilizing said high purity, mat-rVWF composition after step (c). 
     
     
         96 . A method for obtaining a composition comprising a high purity, propeptide depleted mature recombinant rVWF (mat-rVWF), said method comprising the steps of:
 a) providing a solution comprising mat-rVWF/rVWF-PP complex, mat-rVWF, and rVWF propeptide (rVWF-PP);   b) inducing dissociation of said mat-rVWF/rVWF-PP complex in said solution in a) into mat-rVWF and rVWF-PP, wherein said dissociation occurs by disruption of the non-covalently associated mat-rVWF and rVWF-PP, wherein said dissociation is induced by:
 i. addition of at least one chelating agent, or 
 ii. increasing the pH to a pH of at least 7; and 
   c) collecting said mat-rVWF to obtain a high purity, mat-rVWF composition, wherein said high purity, mat-rVWF composition comprises at least 95% mature rVWF and less than 5% rVWF-PP.   
     
     
         97 . A pharmaceutical composition comprising a high purity mat-rVWF generated by the method according to  claim 96 . 
     
     
         98 . A method for obtaining a composition comprising a high purity, propeptide depleted mature recombinant rVWF (high purity mat-rVWF), said method comprising the steps of:
 a) loading a solution comprising pro-rVWF, mat-rVWF/rVWF-PP complex, mat-rVWF, and/or rVWF propeptide (rVWF-PP) onto an anion exchange column, wherein said pro-rVWF, mat-rVWF/rVWF-PP complex, and mat-rVWF are bound to said anion exchange column;   b) washing said anion exchange column in a) containing said bound pro-rVWF, mat-rVWF/rVWF-PP complex, and mat-rVWF with one or more wash buffers;   c) treating said column in b) comprising the bound pro-rVWF, mat-rVWF/rVWF-PP complex, and mat-rVWF with furin, wherein said furin cleaves said pro-rVWF into mat-rVWF and rVWF-PP;   d) eluting said bound pro-rVWF, mat-rVWF/rVWF-PP complex, and mat-rVWF from the column in c) with an elution buffer, wherein said elution buffer induces dissociation of said rVWF-PP from mat-rVWF non-covalently associated with said rVWF-PP, and wherein said dissociation is induced by:
 i. addition of at least one chelating agent into said elution buffer, or 
 ii. increasing the pH of said elution buffer to a pH of at least 7; and 
   e) collecting said mat-rVWF separately from said rVWF-PP to obtain a high purity mat-rVWF composition, wherein said high purity mat-rVWF composition comprises at least 95% mature rVWF and less than 5% rVWF-PP.   
     
     
         99 . A pharmaceutical composition comprising a high purity mat-rVWF generated by the method according to  claim 98  and a pharmaceutically acceptable buffer. 
     
     
         100 . A method for obtaining a composition comprising a high purity, propeptide depleted mature recombinant rVWF (mat-rVWF), said method comprising the steps of:
 a) loading a solution comprising mat-rVWF/rVWF-PP complex, mat-rVWF, and rVWF propeptide (rVWF-PP) onto a size exclusion column;   b) washing said size exclusion column with a buffer, thereby dissociating said mat-rVWF/rVWF-PP complex in said solution in a) into mat-rVWF and rVWF-PP, wherein said dissociation occurs by disruption of the non-covalently associated mat-rVWF and rVWF-PP,