Method for producing insulin analogs having a dibasic B chain terminus

A method for preparing a functional insulin analog or a derivative thereof, wherein the insulin analog is characterized by the general formula I:

• wherein
• (A1-A5) are the amino acid residues in positions A1 to A5 of the A chain of human insulin or animal insulin,
• (A12-A19) are the amino acid residues in positions A12 to A19 of the A chain of human insulin or animal insulin, A21 is a naturally occurring amino acid residue,
• (B8-B18) are the amino acid residues in positions B8 to B18 of the B chain of human insulin or animal insulin,
• (B20-B26) are the amino acid residues in positions B20 to B26 of the B chain of human insulin or animal insulin,
• (A8-A10) are the amino acid residues in positions A8 to A10 of the A chain of human insulin or animal insulin,
• B30 is a chemical bond or a naturally occurring amino acid residue,
• B1 is a chemical bond or a naturally occurring amino acid residue,
• B3 is a naturally occurring amino acid residue,
• each of B27, B28 and B29 is a naturally occurring amino acid residue,
• R1 is an amino group or one to three naturally occurring amino acid residues,
• R2 is a carboxy group or one to three naturally occurring amino acid residues,
• R3 is an amino group or one to three naturally occurring amino acid residues,
• R4 is a basic amino acid selected from the group consisting of Arg and Lys, and
• R5 is one or two basic amino acid residues whose C terminus is either free or amidated,
• said method comprising:
• adding a naturally occurring, basic amino acid which is amidated or protected C-terminally with a protective group, onto an initial insulin analog or a derivative thereof whose C-terminal amino acid of the A and/or B chain is selected from a group comprising naturally occurring, basic amino acids or analogs or derivatives thereof, onto one of said C-terminal amino acids in the presence of an enzyme having the biological activity of trypsin in the presence of an organic solvent under conditions such that said enzyme catalyzes ligation to add said basic amino acid onto one of said C-terminal amino acids;
• purifying the resulting modified insulin analog, and optionally
• cleaving the amide group or C-terminal protective group of the added amino acid or of the added peptide.