Novo Nordisk A/s

Per Nørgaard

Svend Havelund

Per Balschmidt

Frantisek Hubalek

Svend Ludvigsen

Peter Kresten Nielsen

Asser Sloth Andersen

Ulla Ribel-Madsen

Inger Lautrup-Larsen

The present invention relates to novel insulin analogues exhibiting resistance towards protease, wherein at least two amino acids are substituted and/or deleted relative to the parent insulin molecule. A method for the preparation of such insulin analogues, insulin preparations containing the insulin analogues of the invention and a method of treating diabetes mellitus using these insulin analogues is also provided.

Protease resistant insulin analogues

The invention is related to a method for making mature human insulin or an analogue thereof by culturing a fungi cell comprising a DNA sequence encoding a precursor for human insulin or an analogue of human insulin which precursor comprises the B-chain of human insulin or an analogue thereof, the A-chain of human insulin or an analogue thereof and a C-peptide linking the B-chain and the A-chain together thereof, wherein the C-peptide comprises at least one Kex2 cleavage site and an amino acid sequence attached at one end to the C-terminal amino acid residue in the B-chain and at the other end to the Kex2 site which amino acid sequence will facilitate a more efficient Kex2 cleavage within the fungi cell. The C-terminal extension of the B-chain may furthermore be capable of subsequently being cleaved off from the C-terminal amino acid residue in the B-chain by means of a carboxypeptidase activity either within the fungi cell or subsequently in the culture medium.

Method for making maturated insulin polypeptides in a fungal cell

The invention is related to a method for making an activated carboxypeptidase in a fungi cell comprising introducing a DNA sequence encoding a proform of the carboxypeptidase wherein a Kex2 site has been introduced in the prosequence of the carboxypeptidase, culturing the fungi cell under conditions suitable for expression of the procarboxypeptidase and cleaving off the prosequence within the cell to liberate the free active form of the carboxypeptidase. The invention is also related to methods for making mature human insulin and human insulin analogues by use of the activated carboxypeptidase enzyme.

Method of making activated carboxypeptidases

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Humlebæk, Denmark

Per Nørgaard