Method for the production of conjugates of insulin-like growth factor-1 and poly(ethylene glycol)

The method of preparing lysine-PEGylated IGF-I or lysine-PEGylated IGF-I variant comprising

• (A) cultivating a prokaryotic host cell comprising an expression vector containing a nucleic acid encoding a fusion protein comprising insulin-like growth factor I (IGF-I) (SEQ ID NO:
• 1) or an IGF-I variant N-terminally linked to the C-terminus of a propeptide, said C-terminus of said propeptide comprising the amino acid sequence Y-Pro, wherein Y is selected from the group consisting of Pro;
• Pro-Ala;
• Pro-Gly;
• Pro-Thr;
• Ala-Pro;
• Gly-Pro;
• Thr-Pro;
• Arg-Pro;
• Pro-Arg-Pro;
• Ala-Pro-Arg-Pro (SEQ ID NO:
• 12);
• and Pro-Ala-Pro-Arg-Pro (SEQ ID NO:
• 13) and said variant being a polypeptide which differs from SEQ ID NO:
• 1 in that one or two of the lysine residues at positions 27, 65, or 68 thereof is independently substituted by a polar amino acid selected from the group consisting of cysteine;
• aspartic acid;
• glutamic acid;
• histidine;
• asparagine;
• glutamine;
• arginine;
• serine;
• and threonine;
• and the remainder of the amino acid sequence of said variant is the same as that of SEQ ID NO:
• 1 and causing said cell to express said fusion protein;
• (B) recovering said fusion protein;
• (C) lysine-PEGylating said fusion protein;
• (D) cleaving said lysine-PEGylated fusion protein with IgA protease, and
• (E) recovering said lysine-PEGylated IGF-I or lysine-PEGylated IGF-I variant produced in step (D).