Methods for increasing protein polyethylene glycol (peg) conjugation

Abstract

The present invention relates to highly conjugated proteins and methods for making such proteins. In particular, the present invention relates to methods for linking additional sites to a protein for conjugation with activated polyethylene glycol (PEG) linkers, without denaturing the protein. The invention also relates to highly conjugated proteins with decreased immunogenicity and increased circulating half-life.

1 . A modified protein wherein said modification comprises at least one first polyethylene glycol (PEG) polymer coupled to at least one amino group of said protein and at least one second PEG polymer coupled to a carboxyl group of said protein through a polyamine linker, wherein one amino group of said polyamine linker forms an amide with said carboxyl group of the protein and at least one additional amino group is coupled to said second PEG polymer. 
     
     
         2 . The modified protein of  claim 1  wherein more than one first PEG polymer are coupled to more than one amino group of said protein. 
     
     
         3 . The modified protein of  claim 1  wherein more than one second PEG polymer are coupled to more than one amino group of said polyamine linker. 
     
     
         4 . The modified protein of  claim 2  wherein more than one second PEG polymer are coupled to more than one amino group of said polyamine linker. 
     
     
         5 . The modified protein of  claim 1  wherein the poly functional amine linker is diaminobutane.