Method for preparing a site-specific physiologically active polypeptide conjugate

A method for preparing a site-specific physiologically active polypeptide conjugate, comprising the steps of:

• i) determining a kind and an amount of a first alcohol and a pH range where a target site conjugate exist more than a non-target site conjugate by identifying a ratio of the target site conjugate and non-target site conjugate , depending on the change of the kind and the amount of the first alcohol and pH in a reaction medium, wherein the target site conjugate is a conjugate of a physiologically active polypeptide and a non-peptidyl polymer, said non-peptidyl polymer binding to a target amino acid residue of the physiologically active polypeptide, and wherein the non-target site conjugate is a conjugate of a physiologically active polypeptide and a non-peptidyl polymer, said non-peptidyl polymer binding to an amino acid residue other than the target amino acid residue of the physiologically active polypeptide;
• ii) reacting the physiologically active polypeptide with the non-peptidyl polymer in a reaction medium having the kind and the amount of the first alcohol and the pH range determined in step i);
• and
• iii) isolating and purifying the physiologically active polypeptide conjugate from the reaction mixture of step ii) by ion exchange chromatography using a second alcohol,
• wherein the non-peptidyl polymer is selected form the group consisting of polyethylene glycol, polypropylene glycol, copolymers of ethylene glycol and propylene glycol, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethyl ether, poly(lactic acid), polylactic-glycolic acid, lipid polymers, chitins, hyaluronic acid, and combinations thereof;
• and
• wherein the non-peptidyl polymer has a reactive aldehyde, propionaldehyde or butyraldehyde group at both ends thereof.