Refolding of Recombinant Proteins

Abstract

Processes are provided for recovering and purifying refolded recombinant proteins produced in heterologous host cells, which includes the step of refolding the protein in a high pH buffer.

1 . A process for recovering a refolded recombinant protein from a prokaryotic cell culture, the process comprising the steps of:
 (a) isolating a recombinant protein from the prokaryotic cell culture;   (b) solubilizing said protein in a first buffered solution, pH greater than 9, comprising a first chaotropic agent;   (c) refolding said solubilized protein in a second buffered solution, pH>9 but ≦11, comprising a second chaotropic agent, two or more reducing agents and addition of air or oxygen for such a time and under such conditions that refolding of the recombinant protein occurs; and   (d) recovering said refolded recombinant protein.   
     
     
         2 . The process of  claim 1 , wherein the recombinant protein is a growth factor. 
     
     
         3 . The process of  claim 2 , wherein the growth factor is vascular endothelial growth factor (VEGF). 
     
     
         4 . The process of  claim 3 , wherein the VEGF is VEGF 165 . 
     
     
         5 . The process of  claim 1 , wherein the first and second chaotropic agents are urea. 
     
     
         6 . The process of  claim 1 , wherein the first buffered solution further comprises arginine. 
     
     
         7 . The process of  claim 1 , wherein the second buffered solution further comprises arginine. 
     
     
         8 . The process of  claim 1 , wherein the first buffered solution comprises 1 M Urea, 300 mM arginine, 10 mM CHES, 5 mM EDTA, pH 1, final concentration. 
     
     
         9 . The process of  claim 1 , wherein the first buffered solution comprises 1 M Urea, 300 mM arginine, 10 mM TRIS, 5 mM EDTA, pH 11, final concentration. 
     
     
         10 . The process of  claim 1 , wherein the second buffered solution comprises 1 M Urea, 15 mM cysteine, 2 mM DTT, 100 mM arginine, 10 mM CHES, 5 mM EDTA, pH 10, final concentration. 
     
     
         11 . The process of  claim 1 , wherein the second buffered solution comprises 1 M Urea, 15 mM cysteine, 0.5-2 mM DTT, 100 mM arginine, 10 mM TRIS, 5 mM EDTA, pH 10, final concentration. 
     
     
         12 . The process of  claim 1 , wherein the recombinant protein is incubated in the first buffered solution for at least 1 hour. 
     
     
         13 . The process of  claim 12 , wherein the incubation is performed at 2-40° C. 
     
     
         14 . The process of  claim 1 , wherein the solubilized protein is incubated in the second buffered solution for about 3 to 24 hours. 
     
     
         15 . The process of  claim 14 , wherein the incubation is performed at 2-40° C. 
     
     
         16 . The process of  claim 1 , wherein the two or more reducing agents comprises cysteine and DTT. 
     
     
         17 . The process of  claim 1 , wherein the addition of air or oxygen is provided at ak La =0.001 to 0.1 min −1 . 
     
     
         18 . The process of  claim 1 , further comprising stabilizing the refolded recombinant protein by adding nitrogen. 
     
     
         19 . The process of  claim 1 , wherein said recovery step (d) comprises clarifying the second buffered solution with the recombinant protein and sequentially contacting said refolded recombinant protein to a mixed mode chromatographic support, a cationic chromatographic support, and a first hydrophobic chromatographic support, and selectively eluting the refolded recombinant protein from each support. 
     
     
         20 . The process of  claim 19 , wherein the clarifying step comprises: adding detergent to a final concentration of 1%, adjusting pH to about 8.5-9.5, incubating solution for  1  to  10  hours at 25-30° C., centrifuging the solution; and filtering liquid recovered from the centrifugation step. 
     
     
         21 . The process of  claim 19 , further comprising contacting said refolded recombinant protein to a second hydrophobic chromatographic support or an ion exchange support and selectively eluting the refolded recombinant protein from the support. 
     
     
         22 . The process of  claim 19  or  21 , wherein said first and second hydrophobic interaction chromatographic support is selected from the group consisting of butyl-, propyl-, octyl-, phenyl- and aryl-agarose resins. 
     
     
         23 . A process for recovering a refolded recombinant protein from a prokaryotic cell culture, the method comprising the steps of:
 (a) isolating a recombinant protein from the prokaryotic cell culture;   (b) solubilizing and refolding said protein in a combo buffered solution, pH>9 but ≦11, with addition of air or oxygen; and,   (c) recovering said refolded recombinant protein.   
     
     
         24 . The process of  claim 23 , wherein the recovery step comprises clarifying the combo solution with the recombinant protein and sequentially contacting said refolded recombinant protein to a mixed mode chromatographic support, a cationic chromatographic support, and a first hydrophobic chromatographic support, and selectively eluting the refolded recombinant protein from each support. 
     
     
         25 . The process of  claim 24 , wherein the clarifying step comprises: adding detergent to a final concentration of 1%, adjusting pH to about 8.5-9.5, incubating solution for 1 to 10 hours at 25-30° C., centrifuging the solution; and filtering liquid recovered from the centrifugation step. 
     
     
         26 . The process of  claim 23 , wherein the combo buffered solution comprises 1 M Urea, 15 mM cysteine, 2 mM DTT, 100 mM arginine, 10 mM CHES, 5 mM EDTA, pH 10, final concentration. 
     
     
         27 . The process of  claim 23 , wherein the combo buffered solution comprises 1 M Urea, 15 mM cysteine, 0.5-2 mM DTT, 100 mM arginine, 10 nM TRIS, 5 mM EDTA, pH 10, final concentration. 
     
     
         28 . The process of  claim 23 , wherein the recombinant protein is incubated in the combo buffered solution for about 3 to 24 hours. 
     
     
         29 . The process of  claim 28 , wherein the incubation is performed at 2-40° C. 
     
     
         30 . The process of  claim 24 , further comprising contacting said refolded recombinant protein to a second hydrophobic chromatographic support or an ion exchange support and selectively eluting the refolded recombinant protein from the support. 
     
     
         31 . A method for purifying a recombinant protein, the method comprising the steps of: sequentially contacting a recombinant protein with a mixed mode support, a cationic chromatographic support, a first hydrophobic interaction chromatographic support, and selectively eluting the recombinant protein from each support. 
     
     
         32 . The method of  claim 30 , further comprising contacting the recombinant protein with a second hydrophobic chromatographic support or an ion exchange support, and selectively eluting the recombinant protein from the support.