Aggregate-free protein compositions and methods of preparing same

Abstract

A naturally occurring or recombinant protein, especially a mutein of porcine urate oxidase (uricase), that is essentially free of large aggregates can be rendered substantially non-immunogenic by conjugation with a sufficiently small number of strands of polymer such that the bioactivity of the protein is essentially retained in the conjugate. Such conjugates are unusually well suited for treatment of chronic conditions because they are less likely to induce the formation of antibodies and/or accelerated clearance than are similar conjugates prepared from protein preparations containing traces of large aggregates.

1 - 33 . (cancelled).  
     
     
         34 . A conjugate comprising a therapeutic protein covalently linked to poly(ethylene glycol) or poly(ethylene oxide), wherein the protein in said conjugate is substantially free of aggregates.  
     
     
         35 . The conjugate of  claim 34 , wherein said protein is selected from the group consisting of a naturally occurring protein, a recombinant protein and a mutant protein that has substantially the structure of a naturally occurring protein.  
     
     
         36 . The conjugate of  claim 34 , wherein said protein is a growth factor or a growth hormone.  
     
     
         37 . The conjugate of  claim 34 , wherein said protein is an interferon.  
     
     
         38 . The conjugate of  claim 37 , wherein said interferon is interferon-alpha.  
     
     
         39 . The conjugate of  claim 34 , wherein said protein is an Immunoglobulin G (IgG).  
     
     
         40 . The conjugate of  claim 34 , wherein said poly(ethylene glycol) is monomethoxy poly(ethylene glycol).  
     
     
         41 . The conjugate of  claim 34 , wherein said protein is conjugated to said poly(ethylene glycol) or poly(ethylene oxide) via a linkage selected from the group consisting of a urethane (carbamate) linkage, a secondary amine linkage and an amide linkage.  
     
     
         42 . The conjugate of  claim 34 , wherein said poly(ethylene glycol) or poly(ethylene oxide) has a molecular weight of between about 10 kDa and about 60 kDa.  
     
     
         43 . The conjugate of  claim 34 , wherein the average number of strands of said poly(ethylene glycol) or poly(ethylene oxide) that are covalently linked to said protein is between 2 and 12.  
     
     
         44 . The conjugate of  claim 34 , wherein said poly(ethylene glycol) or poly(ethylene oxide) is linear.  
     
     
         45 . The conjugate of  claim 34 , wherein said poly(ethylene glycol) or poly(ethylene oxide) is branched.  
     
     
         46 . A method for preparing a protein composition that is substantially free of aggregates of said protein, comprising: 
 (a) preparing a solution of a protein in a suitable solvent;    (b) separating said protein solution by a separation process, whereby a plurality of fractions of said protein is obtained;    (c) assessing the level of aggregation of said protein during or following said separation; and    (d) separating the fractions of said protein that contain substantially aggregated protein from those fractions that are substantially free of aggregates of said protein.    
     
     
         47 . The method of  claim 46 , wherein the aggregation of said protein in said individual fractions is assessed by measuring the light scattering of each of said fractions.  
     
     
         48 . The method of  claim 46 , wherein said separation process is selected from the group consisting of ion-exchange chromatography, size-exclusion chromatography and ultrafiltration.  
     
     
         49 . The method of  claim 46 , wherein said protein is selected from the group consisting of a naturally occurring protein, a recombinant protein and a mutant protein that has substantially the structure of a naturally occurring protein.  
     
     
         50 . The method of  claim 46 , wherein said protein is a growth factor or a growth hormone.  
     
     
         51 . The method of  claim 46 , wherein said protein is an interferon.  
     
     
         52 . The method of  claim 51 , wherein said interferon is interferon-alpha.  
     
     
         53 . The method of  claim 46 , wherein said protein is an Immunoglobulin G (IgG).  
     
     
         54 . A method for preparing a conjugate of a therapeutic protein having reduced immunogenicity, comprising: 
 (a) preparing a protein that is substantially free of aggregates of said protein according to the method of  claim 46;  and    (b) covalently linking said protein to one or more poly(ethylene glycol) or poly(ethylene oxide) molecules.    
     
     
         55 . The method of  claim 54 , wherein said poly(ethylene glycol) is monomethoxy poly(ethylene glycol).  
     
     
         56 . The method of  claim 54 , wherein said protein is conjugated to said poly(ethylene glycol) or poly(ethylene oxide) via a linkage selected from the group consisting of a urethane (carbamate) linkage, a secondary amine linkage and an amide linkage.  
     
     
         57 . The method of  claim 54 , wherein said poly(ethylene glycol) or poly(ethylene oxide) has a molecular weight of between about 10 kDa and about 60 kDa.  
     
     
         58 . The method of  claim 54 , wherein the average number of strands of said poly(ethylene glycol) or poly(ethylene oxide) that are covalently linked to said protein is between 2 and 12.  
     
     
         59 . The method of  claim 54 , wherein said poly(ethylene glycol) or poly(ethylene oxide) is linear.  
     
     
         60 . The method of  claim 54 , wherein said poly(ethylene glycol) or poly(ethylene oxide) is branched.  
     
     
         61 . A pharmaceutical composition comprising the conjugate of  claim 34  and one or more pharmaceutically acceptable excipients.  
     
     
         62 . The pharmaceutical composition of  claim 61 , wherein said composition is stabilized by lyophilization and dissolves upon reconstitution to provide a solution suitable for parenteral administration.