Methods of protein purification and recovery

Abstract

Improved methods for purification and recovery of interferon-beta (IFN-β) and compositions comprising substantially monomeric IFN-β are provided. In one purification method, substantially purified IFN-β or variant thereof is precipitated and then dissolved in a guanidine hydrochloride (HCl) solution. Renaturation of the protein occurs by dilution with a suitable buffer. A similar purification method absent the precipitation step is also provided. Following renaturation of the IFN-β, residual guanidine HCl is removed by diafiltration or dialysis with a pharmaceutically acceptable buffer to prepare pharmaceutical compositions comprising substantially monomeric IFN-β.

That which is claimed is:  
     
         1 . A method for preparing an injectable formulation of interferon-beta (IFN-β) comprising: 
 a) preparing a first solution comprising IFN-β, isolating a pool of purified IFN-β from this solution, and precipitating said IFN-β from this pool using an alcohol to form a precipitate;  
 b) dissolving said precipitate in guanidine hydrochloride (HCl) to form a second solution comprising resolubilized denatured IFN-β and guanidine HCl;  
 c) diluting said second solution into a first buffer to obtain a third solution comprising resolubilized renatured IFN-beta and residual guanidine HCl; and  
 d) removing residual guanidine HCl from said third solution by diafiltration or dialysis of said third solution into a second buffer that is pharmaceutically acceptable, whereby said injectable formulation of IFN-β is prepared.  
 
     
     
         2 . The method of  claim 1 , wherein said second buffer contains arginine or sodium chloride.  
     
     
         3 . The method of  claim 1 , wherein said first buffer has a pH of about 5.0 to about 8.0, and wherein said residual guanidine HCl is present in said third solution at a concentration of 1.6 M or less.  
     
     
         4 . The method of  claim 1 , wherein said IFN-β has the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2.  
     
     
         5 . The method of  claim 1 , wherein said IFNβ is glycosylated or unglycosylated.  
     
     
         6 . The method of  claim 1 , wherein said IFNβ is recombinantly produced.  
     
     
         7 . The method of  claim 1 , wherein said IFN-β has at least 80% amino acid sequence identity with the amino acid sequence set forth in SEQ ID NO:1 as calculated using the ALIGN program with a PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.  
     
     
         8 . A method for preparing an injectable formulation of interferon-beta (IFN-β), said method comprising denaturation of IFN-β with guanidine hydrochloride (HCl) followed by renaturation of the IFN-β via dilution into a first buffer to obtain a renatured IFN-β solution comprising residual guanidine HCl, and removing said residual guanidine HCl from said renatured IFN-β solution by diafiltration or dialysis of said renatured IFN-β solution into a second buffer that is pharmaceutically acceptable, whereby said injectable formulation of IFN-β is prepared.  
     
     
         9 . The method of  claim 8 , wherein said first buffer has a pH of about 3.0 to about 5.0, and wherein said residual guanidine HCl is present in said renatured IFN-β solution at a concentration of 1.6 M or less.  
     
     
         10 . The method of  claim 9 , wherein said first buffer has a pH of about 3.0 to about 4.0, and wherein said residual guanidine HCl is present in said renatured IFN-β solution at a concentration of 0.2 M or less.  
     
     
         11 . The method of  claim 10 , wherein said first buffer has a pH of about 3.0, and wherein said residual guanidine HCl is present in said renatured IFN-β solution at a concentration of 0.1 M or less.  
     
     
         12 . The method of  claim 8 , wherein said IFN-β has the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2.  
     
     
         13 . The method of  claim 8 , wherein said IFNβ is glycosylated or unglycosylated.  
     
     
         14 . The method of  claim 8 , wherein said IFN-β is recombinantly produced.  
     
     
         15 . The method of  claim 8 , wherein said IFN-β has at least 80% amino acid sequence identity with the amino acid sequence set forth in SEQ ID NO:1 as calculated using the ALIGN program with a PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.  
     
     
         16 . A method for preparing a composition comprising substantially monomeric interferon-beta (IFN-β), said method comprising: 
 a) preparing a precipitate of substantially purified IFN-β;  
 b) dissolving said precipitate in guanidine hydrochloride (HCl) to obtain a first solution comprising resolubilized denatured IFN-β; and  
 c) renaturing said IFN-β by dilution of said first solution with a buffer solution.  
 
     
     
         17 . The method of  claim 16 , wherein said buffer solution has a pH of about 5.0 to about 8.0.  
     
     
         18 . The method of  claim 16 , wherein said IFN-β has the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2.  
     
     
         19 . The method of  claim 16 , wherein said IFN-β is glycosylated or unglycosylated.  
     
     
         20 . The method of  claim 16 , wherein said IFN-β is recombinantly produced.  
     
     
         21 . The method of  claim 16 , wherein said IFN-β has at least 80% amino acid sequence identity with the amino acid sequence set forth in SEQ ID NO:1 as calculated using the ALIGN program with a PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.  
     
     
         22 . A method for preparing an injectable formulation of interferon-beta (IFN-β), said method comprising: 
 a) obtaining a sample comprising substantially purified IFN-β;  
 b) mixing said sample with guanidine hydrochloride (HCl) to obtain a first solution comprising solubilized denatured IFN-β;  
 c) diluting said first solution into a first buffer to obtain a second solution comprising solubilized renatured IFN-beta and residual guanidine HCl; and  
 d) removing residual guanidine HCl from said second solution by diafiltration or dialysis of said second solution into a second buffer that is pharmaceutically acceptable, whereby said injectable formulation of IFN-β is prepared.  
 
     
     
         23 . The method of  claim 22 , wherein said first buffer has a pH of about 3.0 to about 5.0, and wherein said residual guanidine HCl is present in said second solution at a concentration of 1.6 M or less.  
     
     
         24 . The method of  claim 23 , wherein said first buffer has a pH of about 3.0 to about 4.0, and wherein said residual guanidine HCl is present in said second solution at a concentration of 0.2 M or less.  
     
     
         25 . The method of  claim 24 , wherein said first buffer has a pH of about 3.0, and wherein said residual guanidine HCl is present in said renatured IFN-β solution at a concentration of 0.1 M or less.  
     
     
         26 . The method of  claim 22 , wherein said IFN-β has the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2.  
     
     
         27 . The method of  claim 22 , wherein said IFN-β is glycosylated or unglycosylated.  
     
     
         28 . The method of  claim 22 , wherein said IFN-β is recombinantly produced.  
     
     
         29 . The method of  claim 22 , wherein said IFN-β has at least 80% amino acid sequence identity with the amino acid sequence set forth in SEQ ID NO:1 as calculated using the ALIGN program with a PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.  
     
     
         30 . A method for preparing a composition comprising substantially monomeric interferon-beta (IFN-β), said method comprising: 
 a) preparing a sample comprising substantially purified IFN-β;  
 b) mixing said sample with guanidine hydrochloride (HCl) to obtain a first solution comprising solubilized denatured IFN-β; and  
 c) renaturing said IFN-β by dilution of said first solution with a buffer solution.  
 
     
     
         31 . The method of  claim 30 , wherein said buffer solution has a pH of about 3.0 to about 5.0.  
     
     
         32 . The method of  claim 30 , wherein said IFN-β has the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2.  
     
     
         33 . The method of  claim 30 , wherein said IFN-β is glycosylated or unglycosylated.  
     
     
         34 . The method of  claim 30 , wherein said IFN-β is recombinantly produced.  
     
     
         35 . The method of  claim 30 , wherein said IFN-β has at least 80% amino acid sequence identity with the amino acid sequence set forth in SEQ ID NO:1 as calculated using the ALIGN program with a PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.