PROCESS FOR CORRECTION OF A DISULFIDE MISFOLD IN Fc MOLECULES

Abstract

The present invention concerns a process by which a misfold in an Fc fusion molecule can be prevented or corrected. In one embodiment, the process comprises (a) preparing a pharmacologically active compound comprising an Fc domain; (b) treating the fusion molecule with a copper (II) halide; and (c) isolating the treated fusion molecule. The pharmacologically active compound can be an antibody or a fusion molecule comprising a pharmacologically active domain and an Fc domain. The preferred copper (II) halide is CuCl 2 . The preferred concentration thereof is at least about 10 mM for fusion molecules prepared in E. coli ; at least about 30 mM for fusion molecules prepared in CHO cells. The process can be employed with any number of pharmacologically active domains. Preferred pharmacologically active domains include OPG proteins, leptin proteins, soluble portions of TNF receptors (e.g., wherein the fusion molecule is etanercept), IL-1ra proteins, and TPO-mimetic peptides. The Fc domain preferably has a human sequence, with an Fc sequence derived from IgG1 most preferred. An exemplary Fc sequence is shown in FIG. 5 hereinafter.

1 . A process for preparing a pharmacologically active compound, which comprises:
 (a) preparing a fusion molecule comprising an Fc domain and a pharmacologically active compound;   (b) treating the fusion molecule with guanidine HCl at a concentration of at least about 4 M;   (c) increasing the pH to about 8.5; and   (d) isolating the treated fusion molecule.   
     
     
         2 . The process of  claim 1 , wherein the fusion protein is prepared in  E. coli.    
     
     
         3 . The process of  claim 1 , wherein the fusion protein is prepared in CHO cells. 
     
     
         4 . The process of  claim 1 , wherein the pharmacologically active domain comprises the sequence of an OPG protein. 
     
     
         5 . The process of  claim 1 , wherein the pharmacologically active domain comprises the sequence of a leptin protein. 
     
     
         6 . The process of  claim 1 , wherein the pharmacologically active domain comprises the sequence of a TNF-alpha inhibitor. 
     
     
         7 . The process of  claim 1 , wherein the pharmacologically active domain comprises the sequence of an IL-1 inhibitor. 
     
     
         8 . The process of  claim 1 , wherein the pharmacologically active domain comprises the sequence of an IL-1ra protein. 
     
     
         9 . The process of  claim 1 , wherein the pharmacologically active domain comprises the sequence of a TPO-mimetic peptide. 
     
     
         10 . The process of  claim 9 , wherein TPO-mimetic peptide comprises the sequence IEGPTLRQWLAARA (SEQ ID NO:7). 
     
     
         11 . The process of  claim 10 , wherein TPO-mimetic peptide comprises the sequence GGGGG-IEGPTLRQWLAARA-GGGGGGGG-IEGPTLRQWLAARA (SEQ ID NO:32). 
     
     
         12 . The process of  claim 11 , wherein the fusion protein has the sequence of SEQ ID NO:35. 
     
     
         13 . The process of  claim 10 , wherein the fusion protein is prepared in  E. coli.    
     
     
         14 . The process of  claim 12 , wherein the fusion protein is prepared in  E. coli.    
     
     
         15 . The process of  claim 1 , wherein the Fc domain is an IgG1 Fc domain. 
     
     
         16 . The process of  claim 1 , wherein the Fc domain comprises the sequence of SEQ ID NO: 2.