Novo Nordisk A/s

Novo Industri A/s

Jan Markussen

Ib Jonassen

Svend Havelund

Per Balschmidt

Thomas Hoeg-Jensen

Niels Christian Kaarsholm

Michi Egel-Mitani

Asser Sloth Andersen

Ivan Verner Diers

Jakob Brandt

Per Hertz Hansen

Peter Kurtzhals

Kjeld Norris

Thomas Børglum Kjeldsen

Thomas Borglum Kjeldsen

John Halstrom

Lars Thim

Behrend Friedrich Lundt

Frederik Christian Gronvald

Karin D Jorgensen

Soren Ostergaard

Alister J Moody

Lauge Schaffer

Mogens T Hansen

John Halstro Slashedm

Liselotte Langkjaer

Thomas Høeg-Jensen

Gustav Ammerer

Hans O Voigt

Niels Fiil

Signe Ridderberg

Nils L Johansen

Erik K Frandsen

Insulins to which there is connected an amino acid oligomer have satisfactory properties.

Peptide extended insulins

The present invention related to a method of producing polypeptides in transformed host cells by expressing a precursor molecule of the desired polypeptide which are to be acylated in a subsequent in vitro step. The invention is also related to DNA-sequences, vectors and transformed host cells for use in the claimed method. Further, the present invention is related to certain precursors of the desired polypeptides and certain acylation methods. The invention provides a method for making polypeptides being preferentially acylated in certain lysine &#x3b5;-amino groups.

Method for making acylated polypeptides

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 &#x3b5;-amino group in Lysand 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.

Processes for making acylated insulin

Insulin derivatives having a built-in glucose sensor, capable of delivering insulin from a depot as a function of the glucose concentration in the surrounding medium (e.g. tissue), such that the rate of insulin release from the depot increases with an increased glucose concentration and decreases with a decreased glucose concentration.

Glucose dependent release of insulin from glucose sensing insulin derivatives

The present invention relates to insulin derivatives having a built-in glucose sensor and a polyol moiety, capable to deliver insulin from an injected depot of an insulin derivative according to the invention as a function of the glucose concentration in the surrounding tissue.

Glucose-dependent insulins

The present invention relates to insulin derivatives in which a lipophilic group having from 12 to 40 carbon atoms is attached to the &#x3b1;-amino group of the N-terminal amino acid in the B-chain or to the carboxy group of the C-terminal amino acid in the B-chain have a protracted profile of action.

Insulin derivatives

The present invention relates to protracted human insulin derivatives in which the A21 and the B3 amino acid residues are, independently, any amino acid residue which can be coded for by the genetic code except Lys, Arg and Cys; Phemay be deleted; the B30 amino acid residue is (a) a non-codable, lipophilic amino acid having from 10 to 24 carbon atoms, in which case an acyl group of a carboxylic acid with up to 5 carbon atoms is bound to the &#x3b5;-amino group of Lys; or (b) the B30 amino acid residue is deleted or is any amino acid residue which can be coded for by the genetic code except Lys, Arg and Cys, in any of which cases the &#x3b5;-amino group of Lyshas a lipophilic substituent; and any Zncomplexes thereof with the proviso that when B30 is Thr or Ala and A21 and B3 are both Asn, and Pheis present, then the insulin derivative is always present as a Zncomplex.

Acylated insulin

The present invention relates to insulin derivatives in which a lipophilic group having from 12 to 40 carbon atoms is attached to the &agr;-amino group of the N-terminal amino acid in the B-chain or to the carboxy group of the C-terminal amino acid in the B-chain have a protracted profile of action.

The present invention relates to protracted human insulin derivatives in which the A21 and the B3 amino acid residues are, independently, any amino acid residue which can be coded for by the genetic code except Lys, Arg and Cys; Phe.sup.B1 may be deleted; the B30 amino acid residue is (a) a non-codable, lipophilic amino acid having from 10 to 24 carbon atoms, in which case an acyl group of a carboxylic acid with up to 5 carbon atoms is bound to the .epsilon.-amino group of Lys.sup.B29 ; or (b) the B30 amino acid residue is deleted or is any amino acid residue which can be coded for by the genetic code except Lys, Arg and Cys, in any of which cases the .epsilon.-amino group of Lys.sup.B29 has a lipophilic substituent; and any Zn.sup.2+ complexes thereof with the proviso that when B30 is Thr or Ala and A21 and B3 are both Asn, and Phe.sup.B1 is present, then the insulin derivative is always present as a Zn.sup.2+ complex.

The present invention relates to human insulin derivatives having a protracted profile of action in which the A21 and B3 amino acid residues are, independently, any amino acid residue which can be coded for by the genetic code except Lys, Arg and Cys; Phe.sup.B1 may be deleted; the B30 amino acid residue is (a) a non-codable, lipophilic amino acid having from 10 to 24 carbon atoms in which case an acyl group of a carboxylic acid with up to 5 carbon atoms is bound to the E-amino group of Lys.sup.B29 ; (b) any amino acid residue which can be coded by the genetic code except Lys, Arg and Cys, in which case a lipophilic substituent is bound to the E-amino group of Lys.sup.B29 ; or (c) deleted, in which case a lipophilic substituent is bound to the E-amino group of LyS.sup.B29 ; and any Zn.sup.2+ complexes thereof; provided that when the B30 amino acid residue is Thr or Ala, the A21 and B3 amino acid residues are both Asn and Phe.sup.B1 is present, then the insulin derivative is a Zn.sup.2 + complex.

Protein purification method

Insulin analogs characterized by amino acid residue at A21 other than Asn, with a resulting improvement in stability of insulin solutions at acid pH levels. Some insulin analogs may also have amino acid residue changes elsewhere so that these insulin analogs exhibit at least about one charge more than human insulin at a pH value of 7, with preferred substitutions being made for the glutamic acid residues at A4, A17, B13, B21 and/or a basic amino acid residue being substituted at B27. Also contemplated is optional blocking the C-terminal carboxyl group of the B-chain with an amido or ester residue.

Novel insulin peptides

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Herlev, Denmark

Jan Markussen