Methods for maintaining pegylation of polypeptides

Abstract

Described herein are methods for maintaining PEGylation and inhibiting dePEGylation of polypeptides, such as fibronectin-based scaffold proteins, during storage.

What is claimed is: 
     
         1 . A composition comprising a polypeptide conjugated to a PEG moiety through a maleimide linker, wherein the composition has a pH between pH 2.0 and pH 6.0. 
     
     
         2 - 3 . (canceled) 
     
     
         4 . The composition of claim  2 , wherein less than 5% of the maleimide linkers are cleaved over a four week storage period. 
     
     
         5 . The composition of  claim 4 , wherein the polypeptide comprises a tenth fibronectin type III ( 10 Fn3) domain having an altered amino acid sequence relative to the wild-type sequence that binds to a target molecule with a K D  of less than 500 nM. 
     
     
         6 . (canceled) 
     
     
         7 . The composition of  claim 6 , wherein the  10 Fn3 domain does not contain a DK sequence in the C-terminal tail region. 
     
     
         8 . The composition of  claim 7 , wherein the C-terminal tail of the  10 Fn3 domain comprises a cysteine residue, to which the PEG moiety is linked. 
     
     
         9 - 13 . (canceled) 
     
     
         14 . The composition of  claim 8 , wherein the maleimide linker forms a thioether bond with a sulfhydryl group on the polypeptide. 
     
     
         15 - 17 . (canceled) 
     
     
         18 . A method for maintaining PEGylation of polypeptides during storage, comprising storing the polypeptides in a solution buffered to a pH between pH 2.0 and pH 6.0, wherein the polypeptides are conjugated to a polyethylene glycol (PEG) moiety through a maleimide linker, and wherein less than 5% of the maleimide linkers are cleaved over a four week storage period. 
     
     
         19 . The method of  claim 18 , wherein less than 5% of the maleimide linkers are cleaved over an eight week storage period. 
     
     
         20 - 22 . (canceled) 
     
     
         23 . The method of  claim 18 , wherein the polypeptides comprise a tenth fibronectin type III ( 10 Fn3) domain having an altered amino acid sequence relative to the wild-type sequence that binds to a target molecule with a K D  of less than 500 nM. 
     
     
         24 . The method of  claim 23 , wherein the  10 Fn3 domain does not contain a DK sequence in the C-terminal tail region. 
     
     
         25 . The method of  claim 24 , wherein the C-terminal tail of the  10 Fn3 domain comprises a cysteine residue. 
     
     
         26 - 29 . (canceled) 
     
     
         30 . The method of  claim 18 , wherein the maleimide linker forms a thioether bond with a sulfhydryl group on the polypeptides. 
     
     
         31 . The method of  claim 24 , wherein the pH of the solution is between 3.0 and 4.0. 
     
     
         32 . The method of  claim 24 , wherein the pH of the solution is between 3.5 and 4.5. 
     
     
         33 . The method of  claim 24 , wherein the pH of the solution is between 4.0 and 5.0. 
     
     
         34 . The method of  claim 24 , wherein the pH of the solution is between 4.5 and 5.5. 
     
     
         35 . The method of  claim 24 , wherein the pH of the solution is between 5.0 and 6.0. 
     
     
         36 . The method of  claim 24 , wherein the concentration of the PEGylated polypeptides in the solution is from 1-10 mg/mL. 
     
     
         37 . The method of  claim 24 , wherein the PEGylated polypeptides are stored at a temperature from 4° C. to 30° C. 
     
     
         38 . A method for improving the stability of PEGylated polypeptides in a solution, wherein the polypeptides are conjugated to a polyethylene glycol (PEG) moiety through a maleimide linker, comprising the steps of:
 (a) determining the amount of PEG cleaved from the polypeptides over a fixed storage period in the solution; and   (b) if PEG is cleaved from more than 5% of the polypeptides over the storage period, reformulating the PEGylated polypeptides in a storage solution buffered to a pH of between 2.0-6.0.