Processes for making acylated insulin
AssigneeNovo Nordisk A/S
InventorsThomas Børglum Kjeldsen, Jan Markussen
Life of this patent
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Abstract
A method is provided which allows high yields of acylated insulin. The method comprises expressing a singe-chain insulin precursor, preferably in yeast, cleaving the single-chain insulin precursor with a suitable protease which will open the peptide bond between the C-terminal amino group in the B-chain and a connecting peptide connecting the B chain with the A-chain, acylating the two-chain insulin intermediate in the ε-amino group in LysB29 and subjecting the acylated two-chain insulin intermediate to a proteolytic enzyme which will cleave of the N-terminal extension on the B- and A-chains on the precursor molecule.
Claims16
A method for making an acylated insulin or an insulin analogue, said method comprising:
- a) acylating an insulin intermediate with an acylating agent in a solvent, wherein said insulin intermediate has the formula:
- wherein the X3—X4 sequence is selected from the group of sequences consisting of Asp-Pro, Glu-Pro, Asp-Glu, Asp-Gly and Glu-Gly;
- and the X1—X2 sequence is selected from the group of sequences consisting of Asp-Asp-Gly-Asp-Pro (amino acids 1-5 of SEQ ID NO:1), Glu-Glu-Gly-Glu-Pro (amino acids 1-5 of SEQ ID NO:2), Asp-Asp-Gly-Asp-Gly (amino acids 1-5 of SEQ ID NO:3) and Glu-Glu-Gly-Glu-Gly (amino acids 1-5 of SEQ ID NO:4), B-chain is the B-chain of human insulin or an analogue thereof, and A-chain is the A-chain of human insulin or an analogue thereof, at the C-terminal lysine amino group in the B-chain, and b) cleaving the peptide bond between Arg and B1 and between Arg and A1 in the acylated intermediate by trypsin or a trypsin like protease to produce an acylated insulin or insulin analogue.
Dependencies are inferred from the claim text.