Substantially pure histidine-linked protein polymer conjugates

DETAILED DESCRIPTION In one aspect, the present invention includes substantially pure protein-polymer conjugates.
The conjugates include a protein, such as an alpha interferon, covalently conjugated to a polymer, such as a polyethylene glycol, at a histidine (His) residue of the protein.
In the case of an alpha interferon, the histidine is preferably the histidine 34.
Preferably, the alpha interferon is interferon .
alpha.
2b and the conjugates contain about one polymer strand per alpha interferon molecule.
Histidine-linked mono-polymer conjugates of other proteins, such as IL-10, are also included as part of the invention.
Compositions containing the preferred mono-polymer His-linked conjugates may also contain minor amounts of other mono-PEG-protein species, if desired.
In another embodiment of the invention, methods of preparing substantially pure protein-polymer conjugates are provided.
In particular, the methods are directed to preparing the protein-histidine residue linked polymer-conjugates.
The methods include forming a plurality of protein-polymer conjugate species or positional isomers by reacting a protein such as alpha interferon, with a sufficient amount of suitably activated polymer under conditions sufficient to facilitate covalent attachment of protein molecules to activated polymer strands and thereafter substantially isolating the conjugated species or positional isomers in which the His linkage between the protein and polymer is established from the remaining conjugate species.
In one preferred aspect of this embodiment, the activated polymer is a benzotriazole carbonate-activated polymer.
In an alternative aspect, the activated polymer is an oxycarbonyl-oxy-N-dicarboximide-activated polymer such as succinimidyl carbonate (SC-PEG).
These activated polymers allow the artisan to form a reaction pool in which a substantial portion of the conjugates include the polymer strand covalently linked to a histidine residue on the alpha interferon rather than on a lysine residue or N-terminus