Process for isolation and purification of a target protein free of prion protein (prpsc)

Abstract

A process for isolation and purification of a target protein by chromatography wherein the chromatography removes or depletes prions (PrP Sc ), comprising the steps of contacting a potentially PrP SC -contaminated sample comprising a target protein with a multimodal chromatographic material; setting buffer conditions so that the target protein is bound to the multimodal chromatographic material and whereas PrP SC is not binding to the multimodal chromatographic material; followed by elution of the target protein.

1 - 10 . (canceled) 
     
     
         11 : A chromatography process for decontaminating a target protein of infectious prion proteins (PrP Sc ) comprising the steps of
 contacting a potentially PrP Sc -contaminated sample comprising a target protein with a negatively charged 2-(benzoylamino)butanoic acid ligand multimodal chromatographic material,   setting buffer conditions so that the target protein binds and the PrP Sc  do not bind to the negatively charged 2-(benzoylamino)butanoic acid ligand, and then   eluting the target protein from the negatively charged 2-(benzoylamino)butanoic acid ligand,   
       to obtain a PrP Sc -decontaminated fraction containing the target protein. 
     
     
         12 : The process according to  claim 11  wherein the target protein is eluted with an elution buffer by
 increasing or decreasing the ionic strength of the elution buffer, 
 adding one or more alcohols to the elution buffer, and/or 
 increasing or decreasing the pH of the elution buffer. 
 
     
     
         13 : The method of  claim 11  wherein the PrP Sc  removal value in the fraction containing the target protein is >1 to 4 lg(10), as calculated from the amount which was initially applied to the resin. 
     
     
         14 : The method of  claim 11  wherein the PrP Sc  analytical value in the fraction containing the target protein is below detection limit of the prion Western blot assay. 
     
     
         15 : The method of  claim 11  wherein the step of setting buffer conditions comprises the following steps in sequence:
 i) employing a loading and equilibration buffer containing a solvent and/or a non-ionic detergent; and 
 ii) employing one or more wash buffers selected from the group consisting of a wash buffer without a solvent and/or a non-ionic detergent, a wash buffer containing an alcohol and/or an amino acid, a wash buffer containing a high salt concentration, and a wash buffer containing a low salt concentration; and 
 
       wherein the elution buffer contains an alcohol and a high salt concentration. 
     
     
         16 : The method of  claim 11  wherein the step of setting buffer conditions comprises the following steps in sequence:
 i) employing a loading and an equilibration buffer containing a solvent and/or a non-ionic detergent; and 
 ii) employing one or more wash buffers selected from the group consisting of a first wash buffer without a solvent and a non-ionic detergent, a second wash buffer containing an alcohol and an amino acid, a third wash buffer containing a high salt concentration, and a fourth wash buffer containing a low salt concentration; and 
 
       wherein the elution buffer contains an alcohol and a high salt concentration. 
     
     
         17 : The method of  claim 18  wherein:
 i) the loading and equilibration buffer contains tri-n-butylphosphate and/or Triton x-100 in a concentration ranging of from about 0.1 to about 10% (w/w); 
 ii) the second wash buffer contains ethylene glycol and/or lysine/arginine ranging of from about 5 to about 30% (w/w) of ethylene glycol and of from 0.2 to about 1.5 M lysine/arginine; 
 iii) the third wash buffer contains sodium chloride in a concentration ranging of from about 0.5 to about 4 M, in particular of from about 0.5 to about 1.5 M; 
 iv) the fourth wash buffer contains sodium chloride in concentration ranging of from about 0.01 to about 0.2, in particular of from 0.01 to about 0.1 M; 
 v) the elution buffer contains ethylene glycol and/or sodium chloride ranging in concentration of from about 25 to about 75% (w/w), in particular of from about 25 to about 50% of ethylene glycol and from about 0.5 to about 4 M NaCl. 
 
     
     
         18 : The PrP Sc -decontaminated fraction obtained by the method of  claim 11 . 
     
     
         19 : The PrP Sc -decontaminated fraction according to  claim 18  wherein the target protein comprises one or more plasma proteins, peptide hormones, growth factors, cytokines, and polyclonal immunoglobulins proteins, plasma proteins selected from human and animal blood clotting factors including fibrinogen, prothrombin, thrombin, prothrombin complex, FX, FXa, FIX, FIXa, FVII, FVIIa, FXI, FXIa, FXII, FXIIa, FXIII and FXIIIa, von Willebrandt factor, transport proteins including albumin, transferrin, ceruloplasmin, haptoglobin, hemoglobulin and hemopexin, protease inhibitors including β-antithrombin, α-antithrombin, α2-macroglobulin, Cl-inhibitor, tissue factor pathway inhibitor (TFPI), heparin cofactor II, protein C inhibitor (PAI-3), Protein C and Protein S, α-1 esterase inhibitor proteins, α-1 anti-trypsin, antiangionetic proteins including latent-antithrombin, highly glycosylated proteins including α-1-acid glycoprotein, antichymotrypsin, inter-α-trypsin inhibitor, α-2-HS glycoprotein and C-reactive protein and other proteins including histidine-rich glycoprotein, mannmman binding lectin, C4-binding protein, fibronectin, GC-globulin, plasminogen, blood factors such as erythrmopoeitin, interferon, tumor factors, tPA, γCSF.