Semi-recombinant preparation of GLP-1 analogues

Patent metadata

Field	Value
Publication number	US-9732137-B2
Application number	US-81093408-A
Country	US
Kind code	B2
Filing date	Dec 22, 2008
Priority date	Dec 28, 2007
Publication date	Aug 15, 2017
Grant date	Aug 15, 2017

Abstract

A semi-recombinant method for the production of GLP-1 analogues and derivatives with non-proteogenic amino acids in the N-terminal part combining the use of recombinant expression techniques and chemical peptide synthesis.

First claim

The invention claimed is:

1. A method for making a derivative of a glucagon-like peptide-1 (GLP-1) analogue comprising one or more non-proteogenic amino acids in the N-terminal part, said method comprising the steps of:
i) culturing a host cell comprising a nucleotide sequence encoding a precursor molecule of said GLP-1 derivative under suitable conditions for expression of said precursor molecule,
ii) separating the expressed precursor molecule from the culture broth,
iii) acylating an epsilon amino group of at least one lysine residue in the expressed precursor molecule with an acylating agent, which is optionally activated, to obtain a precursor molecule derivative, and optionally isolating said precursor molecule derivative, wherein the acylation step takes place in an aqueous solvent mixture and the pH in the reaction mixture is between 9 and 13,
iv) coupling a N-terminal amino acid extension of 1, 2, 3 or 4 amino acids in length comprising 1, 2, 3, or 4 non-proteogenic amino acids to the expressed precursor molecule derivative, and
v) isolating the resulting derivative of a GLP-1 analogue;
wherein the GLP-1 analogue is [Aib8,Arg34]GLP-1-(7-37);
wherein said one or more non-proteogenic amino acids in the N-terminal amino acid extension are moieties which can be incorporated into a peptide via peptide bonds but is not a proteogenic amino acid that is selected from the group consisting of Alanine, Arginine, Asparagine, Aspartic acid, Cysteine, Cystine, Glutamine, Glutamic acid, Glycine, Histidine, Hydroxyproline, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Proline, Serine, Threonine, Tryptophan, Tyrosine, and Valine;
wherein the acylating agent is a carboxylic acid analogue of the general formula selected from the group consisting of A-OH, A-C-D-OH, A-B-C-OH and A-B-C-D-OH, which is optionally activated and/or protected with one or more protection group(s), wherein,
A is selected from the group consisting of

wherein
n is selected from the group consisting of 14, 15, 16 17, 18 and 19;
p is selected from the group consisting of 10, 11, 12, 13 and 14;
d is selected from the group consisting of 0, 1, 2, 3, 4 and 5;
m is selected from the group consisting of 11, 12, 13, 14, 15, 16, and 17;
k is selected from the group consisting of 0, 1, 2, 3, 4, 5, 11 and 27;
m′ is selected from the group consisting of 0, 1, 2, 3, 4, 5 and 6; and
R1 is a generally accepted protection group;
B is selected from the group consisting of

wherein
x is selected from the group consisting of 0, 1, 2, 3 and 4; and
y is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12;
C is selected from the group consisting of

wherein b and e are each independently selected from the group consisting of 0, 1 and 2, and c and f are each independently selected from the group consisting of 0, 1 and 2 with the proviso that b is 1 or 2 when c is 0, or b is 0 when c is 1 or 2, and e is 1 or 2 when f is 0, or e is 0 when f is 1 or 2;
wherein, when one or more free carboxylic acid(s) are present, they are optionally protected with a generally accepted protection group; and
D is selected from the group consisting of

and wherein k is selected from the group consisting of 0, 1, 2, 3, 4, 5, 11 and 27, and m is selected from the group consisting of 0, 1, 2, 3, 4, 5 and 6; and
wherein the resulting GLP-1 derivative is resistant to degradation by dipeptidyl peptidase IV (DPP-IV).

2. The method according to claim 1 , wherein the host cell is selected from a mammalian host cell, an avian host cell, an insect host cell, a plant host cell, a bacterial host cell, a fungal host cell and a yeast host cell.

3. The method according to claim 1 , wherein step (ii) comprises a further step of removing N-terminal and/or C-terminal pro-peptide extensions from the precursor molecule.

4. The method according to claim 1 , wherein R1 is selected from the group consisting of 9H-fluoren-9-ylmethoxycarbonyl (Fmoc), tert-butoxycarbonyl (Boc), and Benzylcarbamate (Cbz).

5. The method according to claim 1 , wherein the acylating agent is of formula A-C-D-OH, and wherein A-C-D- is selected from the group consisting of:

6. The method according to claim 1 , wherein the acylating agent is of formula A-C-D-OH, and wherein A-C-D- is

7. The method according to claim 1 , wherein said an acylating agent comprises one or more protection groups; and wherein said protection groups are optionally removed from the acylating agent once it has been coupled to the precursor molecule.

8. The method according to claim 1 , wherein said acylation step (iii) takes place in an aqueous solvent mixture with a pH between 10 and 12.

9. The method according to claim 1 , wherein the coupling of the N-terminal amino acid extension comprising 1, 2, 3 or 4 non-proteogenic amino acids takes place in an organic polar solvent selected from the group consisting of 1-methylpyrrolidin-2-one, acetonitrile , tetrahydrofuran, dimethylsulfoxide, N,N-dimethlyformamide and N-methylformamide.

10. A method according to claim 1 , wherein the coupling of the N-terminal amino acid extension comprising 1, 2, 3 or 4 non-proteogenic amino acids takes place in an aqueous solvent mixture.

11. A method according to claim 1 , wherein the GLP-1 derivative is N-epsilon26-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxy-heptadecanoylamino)-butyrylamino]ethoxy }ethoxy)acetylamino]-ethoxy }ethoxy)-acetyl][Aib8,Arg34]GLP-1-(7-37) peptide.

Assignees

• Lau Jesper Faergeman
• Andersen Asser Sloth
• Bloch Paw
• Lau Jesper
• Garibay Patrick William
• Kruse Thomas
• Nørby Inga Sig Nielsen
• Jessen Claus Ulrich
• Christensen Caspar
• Norrild Jens Christian
• Novo Nordisk As

Inventors

• Lau Jesper Faergeman
• Andersen Asser Sloth
• Bloch Paw
• Lau Jesper
• Garibay Patrick William
• Kruse Thomas
• Nørby Inga Sig Nielsen
• Jessen Claus Ulrich
• Christensen Caspar
• Norrild Jens Christian

Classifications

• A61P3/10

  for hyperglycaemia, e.g. antidiabetics · CPC title

• C07K1/061

  using protecting groups · CPC title

• C07K1/107

  by chemical modification of precursor peptides · CPC title

• C07K1/1077

  by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids · CPC title

• C07K1/006

  of peptides containing derivatised side chain amino acids · CPC title