High Pressure Treatment of Aggregated Interferons

Abstract

Methods are provided using high pressure to treat aggregated interferons, to reduce the aggregate content of interferon material. In particular, recombinant human interferon-β may be so treated. Multiple strategies may be used in combination to make nonglycosylated IFN-β more amenable to high pressure treatment. When coupled with purification techniques, these strategies singly or in combination provide a low aggregate or substantially aggregate free, biologically active solution. In certain aspects, pharmaceutical compositions containing nonglycosylated interferon having less than about 5 weight percent of protein aggregation are provided.

1 . A pharmaceutical composition comprising a nonglycosylated interferon, said interferon comprising less than about 5 weight percent of protein aggregation. 
     
     
         2 . The composition of  claim 1 , said interferon comprising less than about 2 weight percent of protein aggregation. 
     
     
         3 . The composition of  claim 1 , said interferon comprising less than about 1 weight percent of protein aggregation. 
     
     
         4 . The composition of  claim 1 , said interferon being essentially aggregate free. 
     
     
         5 . The composition of  claim 1 , wherein said interferon is selected from the group consisting of human IFN-α, IFN-β, and IFN-γ and variants thereof. 
     
     
         6 . The composition of  claim 1 , wherein said interferon is human IFN-β or a variant thereof, said composition being aggregate free and having reduced immunogenicity when compared to a human IFN-β comprising aggregates. 
     
     
         7 . The composition of  claim 1 , wherein said interferon is human IFN-β or a variant thereof. 
     
     
         8 . The composition of  claim 5 , wherein said interferon is a variant according to SEQ ID NO. 2. 
     
     
         9 . The composition of  claim 1 , said composition being substantially free of SDS. 
     
     
         10 . The composition of  claim 1 , said composition being substantially free of HSA. 
     
     
         11 - 21 . (canceled) 
     
     
         22 . The composition of  claim 1 , said composition being sufficiently free of aggregates and/or inclusion bodies so that the composition is stable at 4° C. and comprises less than or equal to about 0.5% aggregates after 4 weeks of incubation at 4° C. 
     
     
         23 . The composition of  claim 1 , said composition being sufficiently free of aggregates and/or inclusion bodies so that the composition is stable after 4 weeks of incubation at 40° C. 
     
     
         24 .- 36 . (canceled) 
     
     
         37 . The composition of  claim 1 , further comprising a buffer in an amount sufficient to maintain the pH of the composition between about 3.0 and about 5.0. 
     
     
         38 . The composition of  claim 37 , wherein the buffer is selected from the group consisting of glycine, aspartic acid, succinate salt, citrate salt, formate salt, acetate salt, glutamic acid, histidine, imidazole, and phosphate. 
     
     
         39 . The composition of  claim 38  wherein the buffer is a sodium salt. 
     
     
         40 . The composition of  claim 1 , further comprising a tonicity modifying agent in an amount sufficient to render the composition isotonic with a patient's bodily fluid. 
     
     
         41 . The composition of  claim 40  wherein the tonicity modifying agent is nonionic. 
     
     
         42 . The composition of  claim 40  wherein the tonicity modifying agent is selected from the group consisting of glucose, mannose, arabinose, ribose, fructose, sorbose, xylulose, sucrose, maltose, trehalose, lactose, glycerol, mannitol, xylitol, sorbitol, and combinations thereof. 
     
     
         43 . The composition of  claim 40  wherein the tonicity modifying agent is present at a concentration of about 1% to about 10%. 
     
     
         44 . A pharmaceutical composition comprising:
 a nonglycosylated interferon, said interferon comprising less than about 5 weight percent of protein aggregation;   a buffer; and   a tonicity modifying agent,   
       wherein the pH of the composition is between about 3.0 and about 5.0. 
     
     
         45 . The composition of  claim 44 , wherein the buffer is present at a concentration no greater than about 60 mM. 
     
     
         46 . The composition of  claim 44 , wherein the buffer is present at a concentration from about 15 mM to about 50 mM. 
     
     
         47 . The composition of  claim 44 , wherein the buffer is selected from the group consisting of glycine, aspartic acid, succinate salt, citrate salt, formate salt, acetate salt, glutamic acid, histidine, imidazole, and phosphate. 
     
     
         48 . The composition of  claim 44  wherein the buffer is a sodium salt of succinate, citrate, formate, or acetate. 
     
     
         49 . The composition of  claim 44  wherein the buffer is sodium acetate. 
     
     
         50 . The composition of  claim 44  wherein the tonicity modifying agent is present in an amount sufficient to render the composition isotonic with a patient's bodily fluid. 
     
     
         51 . The composition of  claim 44  wherein the tonicity modifying agent is nonionic. 
     
     
         52 . The composition of  claim 51  wherein the tonicity modifying agent is selected from the group consisting of glucose, mannose, arabinose, ribose, fructose, sorbose, xylulose, sucrose, maltose, trehalose, lactose, glycerol, mannitol, xylitol, sorbitol, and combinations thereof. 
     
     
         53 . The composition of  claim 51  wherein the tonicity modifying agent is present at a concentration of about 1% to about 10%. 
     
     
         54 . The composition of  claim 44  wherein the tonicity modifying agent is trehalose. 
     
     
         55 . The composition of  claim 54  wherein the tonicity modifying agent is present at a concentration of about 8% to about 10%. 
     
     
         56 . A pharmaceutical composition comprising:
 a nonglycosylated interferon, said interferon comprising less than about 5 weight percent of protein aggregation;   about 15 mM to about 50 mM sodium acetate; and   about 8% to about 10% trehalose,   
       wherein the pH of the composition is between about 3.0 and about 5.0. 
     
     
         57 . A pharmaceutical composition comprising an interferon, said interferon prepared according to a method comprising:
 a) solubilizing interferon-containing inclusion bodies;   b) precipitating the solubilized interferon;   c) incorporating the precipitated interferon into a refolding admixture comprising 0.05 weight percent to 5.0 weight percent of a zwitterionic surfactant and having an absence of a chaotrope or denaturing surfactant; and   d) applying greater than 3000 bars of pressure to the refolding admixture incorporating the interferon such that at least a portion of the interferon refolds to form a composition comprising nonglycosylated interferon, wherein the resulting composition comprises less than about 5 weight percent of protein aggregation.   
     
     
         58 . The composition of  claim 7 , wherein said human IFN-β or variant thereof is PEGylated. 
     
     
         59 . The composition of  claim 7 , wherein said human IFN-β or variant thereof constitutes a portion of a fusion protein.