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WO2004085472A1 - Procede de fabrication de precurseurs de l'insuline humaine et insuline humaine - Google Patents

Procede de fabrication de precurseurs de l'insuline humaine et insuline humaine Download PDF

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Publication number
WO2004085472A1
WO2004085472A1 PCT/DK2004/000209 DK2004000209W WO2004085472A1 WO 2004085472 A1 WO2004085472 A1 WO 2004085472A1 DK 2004000209 W DK2004000209 W DK 2004000209W WO 2004085472 A1 WO2004085472 A1 WO 2004085472A1
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Prior art keywords
insulin
precursor
analogue
human
human insulin
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English (en)
Inventor
Thomas Børglum KJELDSEN
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Novo Nordisk AS
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Novo Nordisk AS
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/62—Insulins

Definitions

  • BACKGROUND Yeast organisms produce a number of proteins that have a function outside the cell.
  • secreted proteins Such proteins are referred to as secreted proteins. These secreted proteins are expressed initially inside the cell in a precursor or a pre-form containing a pre-peptide sequence ensuring effective direction (translocation) of the expressed product across the membrane of the endoplasmic reticulum (ER).
  • the pre-peptide normally named a signal peptide, is generally cleaved off from the desired product during translocation.
  • the protein Once entered in the secretory pathway, the protein is transported to the Golgi apparatus. From the Golgi, the protein can follow different routes that lead to compartments such as the cell vacuole or the cell membrane, or it can be routed out of the cell to be secreted to the external medium (Pfeffer et al. (1987) Ann. Rev. Biochem.
  • Insulin is a polypeptide hormone secreted by ⁇ -cells of the pancreas and consists of two polypeptide chains, A and B, which are linked by two inter-chain disulphide bridges. Furthermore, the A-chain features one intra-chain disulphide bridge.
  • the hormone is synthesized as a single-chain precursor proinsulin (preproinsulin) consisting of a prepeptide of 24 amino acid followed by proinsulin containing 86 amino acids, in the configuration: prepeptide - B - Arg Arg - C - Lys Arg -A, in which C is a connecting peptide of 31 amino acids.
  • Arg-Arg and Lys-Arg are cleavage sites for cleavage of the connecting peptide from the A and B chains.
  • the prior art discloses a limited number of insulin precursors which are expressed in either E. coli or Saccharomyces cerevisiae, vide US 5,962,267, WO 95/16708, EP 0055945, EP 0163529, EP 0347845 and EP 0741188.
  • the present invention features novel connecting peptides (C-peptides) which confer an increased production yield of insulin precursor molecules when expressed in a transformed microorganism, in particular yeast.
  • Such insulin precursors can then be converted into human insulin, desB30 human insulin or certain acylated human insulins by one or more suitable, well known conversion steps
  • the connecting peptides of the present invention consist of 3-4 amino acid residues and contain at least one Asp or one Glu.
  • the connecting peptide contains a cleavage site at its C-terminal end enabling in vitro cleavage of the connecting peptide from the A chain.
  • cleavage site may be any convenient cleavage site known in the art, e.g. a Met cleavable by cyanogen bromide; a single basic amino acid residue or a pair of basic amino acid residues (Lys or Arg) cleavable by trypsin or trypsin like proteases; Acromobactor lyticus protease or by a carboxypeptidase protease.
  • the cleavage site enabling cleavage of the connecting peptide from the A-chain is preferably a single basic amino acid residue Lys or Arg, preferably Lys.
  • Cleavage of the connecting peptide from the B chain is enabled by cleavage at the natural Lys B29 amino acid residue in the B chain giving rise to a desB30 insulin precursor. If the insulin precursor is to be converted into human insulin, the B30 Thr amino acid residue can then be added in vitro by well known, enzymatic procedures.
  • the desB30 insulin may also be converted into an acylated insulin analogue as disclosed in US 5,750,497 and US 5,905,140.
  • the present invention is related to insulin precursors or insulin analogue precursors comprising a sequence of formula: B(1 -29) - X r X 2 -X3- Y - A(1 -21 ), wherein X ⁇ is any amino acid except Cys or is a peptide bond, X 2 is Asp or Glu, X 3 is Asp or Glu and Y is a cleavage site; B(1-29) is the human B-chain or an analogue thereof lacking the amino acid in position B(30) and A(1-21) is the human insulin A chain or an analogue thereof.
  • X-i is a peptide bond
  • X 2 is Glu or Asp
  • X 3 is Glu or Asp
  • Y is Lys or Arg.
  • the sequence X 2 -X 3 -Y can be (a) Glu-Asp-Lys, (b) Glu-Glu-Lys, (c) Asp- Asp-Lys, (d) Asp-Glu-Lys, Glu-Asp-Arg, (e) Glu-Glu-Arg, (f) Asp-Asp-Arg, or (f) Asp-Glu-Arg.
  • the present invention is also related to polynucleotide sequences which code for the claimed insulin precursors or insulin analogue precursors.
  • the present invention is related to vectors containing such polynucleotide sequences and to host cells containing such polynucleotide sequences or vectors.
  • the invention in another aspect, relates to a process for producing insulin precursors or insulin analogue precursors in a host cell, said method comprising (i) culturing a host cell comprising a polynucleotide sequence encoding the insulin precursors of the invention under suitable conditions for expression of said precursor and (ii) isolating the insulin precursor from the culture medium.
  • the invention relates to a process for producing human insulin or desB30 human insulin comprising (i) culturing a host cell comprising a polynucleotide sequence encoding an insulin precursor of the invention; (ii) isolating the insulin precursor from the culture medium and (iii) converting the insulin precursor into desBSO human insulin or human insulin by in vitro enzymatic conversion.
  • the invention relates to a process for producing an acylated desBSO human insulin comprising (i) culturing a host cell comprising a polynucleotide sequence encoding an insulin precursor of the invention; (ii) isolating the insulin precursor from the culture medium, (iii) converting the insulin precursor into desBSO human insulin and (iv) converting the desBSO human insulin into an acylated derivate by use of a convenient acylation method.
  • the present invention is related to a process for making human insulin or a human insulin analogue, said method comprising converting an insulin precursor or insulin analogue precursor according as described above into human insulin or a human insulin analogue by suitable in vitro chemical or enzymatic conversion.
  • the host cell is a yeast host cell and in a further embodiment the yeast host cell is selected from to the genus Saccharomyces. In a further embodiment, the yeast host cell is selected from the species Saccharomyces cerevisiae.
  • connecting peptide or “C-peptide” is meant the connection moiety "C” of the B-C-A polypeptide sequence of a single chain preproinsulin-like molecule. Specifically, in the natural insulin chain, the C-peptide connects position 30 of the B chain and position 1 of the A chain.
  • a "mini C-peptide” or “connecting peptide” such as those described herein, connect B29 to A1 and differ in sequence and length from that of the natural C-peptide.
  • desB30 or “B(1-29) is meant a natural insulin B chain lacking the B30 amino acid residue
  • A(1-21) means the natural insulin A chain
  • the mini C-peptide and its amino acid sequence is indicated in the three letter amino acid code.
  • insulin precursor is meant a single-chain polypeptide which by one or more subsequent chemical and/or enzymatic processes can be converted into human insulin or desBSO human insulin.
  • insulin analogue precursor an insulin precursor molecule having one or more mutations, substitutions, deletions and or additions of the A and/or B amino acid chains relative to the human insulin molecule.
  • the insulin analogues are preferably such wherein one or more of the naturally occurring amino acid residues, preferably one, two, or three of them, have been substituted by another codable amino acid residue.
  • the instant invention comprises analogue molecules having position 28 of the B chain altered relative to the natural human insulin molecule.
  • position 28 is modified from the natural Pro residue to one of Asp, Lys, or lie.
  • the natural Pro residue at position B28 is modified to an Asp residue.
  • Lys at position B29 is modified to Pro;
  • Asn at position A21 may be modified to Ala, Gin, Glu, Gly, His, lie, Leu, Met, Ser, Thr, Trp, Tyr orVal, in particular to Gly, Ala, Ser, orThr and preferably to Gly.
  • Asn at position B3 may be modified to Lys.
  • Further non- limiting examples of insulin analogue precursors are des(B30) human insulin, insulin analogues wherein Phe B1 has been deleted; insulin analogues wherein the A-chain and/or the B-chain have an N-terminal extension and insulin analogues wherein the A-chain and/or the B-chain have a C-terminal extension.
  • the present invention features novel mini C-peptides connecting position 29 of the insulin B chain and position 1 of the insulin A chain which significantly increased production yields in a yeast host cell.
  • significantly increased production is meant an increase in secreted amount of the insulin precursor molecule present in the culture supernatant compared to the yield of an insulin precursor with no Gly in the mini C peptide.
  • An “increased” fermentation yield is an absolute number larger than the control; preferably, the increase is 50% or more larger than the control.
  • POT' is the Schizosaccharomyces pombe triose phosphate isomerase gene
  • TPI1 is the S. cerevisiae triose phosphate isomerase gene.
  • leader is meant an amino acid sequence consisting of a pre-peptide (the signal peptide) and a pro-peptide.
  • signal peptide is understood to mean a pre-peptide which is present as an N-terminal sequence on the precursor form of a protein.
  • the function of the signal peptide is to allow the heterologous protein to facilitate translocation into the endoplasmic reticulum.
  • the signal peptide is normally cleaved off in the course of this process.
  • the signal peptide may be heterologous or homologous to the yeast organism producing the protein.
  • a number of signal peptides which may be used with the DNA construct of the invention including yeast aspartic protease 3 (YAP3) signal peptide or any functional analog (Egel-Mitani et al.
  • pro-peptide means a polypeptide sequence whose function is to allow the expressed polypeptide to be directed from the endoplasmic reticulum to the Golgi apparatus and further to a secretory vesicle for secretion into the culture medium (i.e.
  • the pro-peptide may be the yeast ⁇ -factor pro-peptide, vide US 4,546,082 and 4,870,008.
  • the pro-peptide may be a synthetic pro-peptide, which is to say a pro-peptide not found in nature. Suitable synthetic pro-peptides are those disclosed in US 5,395,922; 5,795,746; 5,162,498 and WO 98/32867.
  • the pro- peptide will preferably contain an endopeplidase processing site at the C-terminal end, such as a Lys- Arg sequence or any functional analog thereof.
  • the polynucleotide sequence of the invention may be prepared synthetically by established standard methods, e.g. the phosphoamidite method described by Beaucage et al. (1981) Tetrahedron Letters 22:1859-1869, or the method described by Matthes et al. (1984) EMBO Journal 3:801-805.
  • oligonucleotides are synthesized, for example, in an automatic DNA synthesizer, purified, duplexed and ligated to form the synthetic DNA construct.
  • a currently preferred way of preparing the DNA construct is by polymerase chain reaction (PCR).
  • the polynucleotide sequence of the invention may also be of mixed genomic, cDNA, and synthetic origin.
  • a genomic or cDNA sequence encoding a leader peptide may be joined to a genomic or cDNA sequence encoding the A and B chains, after which the DNA sequence may be modified at a site by inserting synthetic oligonucleotides encoding the desired amino acid sequence for homologous recombination in accordance with well-known procedures or preferably generating the desired sequence by PCR using suitable oligonucleotides.
  • the invention encompasses a vector which is capable of replicating in the selected microorganism or cell line and which carries a polynucleotide sequence encoding the insulin precursors of the invention.
  • the recombinant vector may be an autonomously replicating vector, i.e., a vector which exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extra-chromosomal element, a mini-chromosome, or an artificial chromosome.
  • the vector may contain any means for assuring self-replication.
  • the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated.
  • a single vector or plasmid or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell, or a transposon may be used.
  • the vectors may be linear or closed circular plasmids and will preferably contain an element(s) that permits stable integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.
  • the recombinant expression vector is capable of replicating in yeast organisms.
  • sequences which enable the vector to replicate in yeast are the yeast plasmid 2 ⁇ m replication genes REP 1-3 and origin of replication.
  • the vectors of the present invention preferably contain one or more selectable markers which permit easy selection of transformed cells.
  • a selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.
  • Examples of bacterial selectable markers are the dal genes from Bacillus subtilis or Bacillus licheniformis, or markers which confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol ortetracycline resistance.
  • Selectable markers for use in a filamentous fungal host cell include amdS (acetamidase), argB (omithine carbamoyltransferase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate adenyltransferase) and trpC (anthranilate synthase.
  • Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1 , and URA3.
  • a preferred selectable marker for yeast is the Schizosaccharomyces pompe TPI gene (Russell (1985) Gene 40:125-130).
  • the polynucleotide sequence is operably connected to a suitable promoter sequence.
  • the promoter may be any nucleic acid sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.
  • suitable promoters for directing the transcription in a bacterial host cell are the promoters obtained from the E. coli lac operon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilis levansucrase gene (sacB), Bacillus licheniformis alpha- amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), and Bacillus licheniformis penicillinase gene (penP).
  • dagA Streptomyces coelicolor agarase gene
  • sacB Bacillus subtilis levansucrase gene
  • amyL Bacillus stearothermophilus maltogenic amylase gene
  • amyQ Bacillus amyloliquefaciens alpha-amylase gene
  • penP Bacillus lichen
  • promoters for directing the transcription in a filamentous fungal host cell are promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha- amylase, and Aspergillus niger acid stable alpha-amylase.
  • useful promoters are the Saccharomyces cerevisiae Ma1, TPI, ADH or PGK promoters.
  • the polynucleotide construct of the invention will also typically be operably connected to a suitable terminator.
  • a suitable terminator is the TPI terminator (Alber et al. (1982) J. Mol. Appl. Genet. 1: 19-434).
  • TPI terminator Alber et al. (1982) J. Mol. Appl. Genet. 1: 19-434.
  • the vector may be constructed either by first preparing a DNA construct containing the entire DNA sequence of the invention, and subsequently inserting this fragment into a suitable expression vector, or by sequentially inserting DNA fragments containing genetic information for the individual elements (such as the signal, pro-peptide, mini C-peptide, A and B chains) followed by ligation.
  • the present invention also relates to recombinant host cells, comprising a polynucleotide sequence encoding the insulin precursors of the invention.
  • a vector comprising such polynucleotide sequence is introduced into the host cell so that the vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector as described earlier.
  • the term "host cell” encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.
  • the host cell may be a unicellular microorganism, e.g., a prokaryote, or a non-unicellular microorganism, e.g., a eukaryote.
  • Useful unicellular cells are bacterial cells such as gram positive bacteria including, but not limited to, a Bacillus cell, Streptomyces cell, or gram negative bacteria such as E. coli and Pseudomonas sp.
  • Eukaryote cells may be mammalian, insect, plant, or fungal cells.
  • the host cell is a yeast cell.
  • the yeast organism used in the process of the invention may be any suitable yeast organism which, on cultivation, produces large amounts of the insulin precursor and insulin precursor analogs of the invention.
  • yeast organisms are strains selected from the yeast species Saccharomyces cerevisiae, Saccharomyces kluyveri, Schizosaccharomyces pombe, Sacchoromyces uvarum, Kluyveromyces lactis, Hansenula polymorpha, Pichia pastoris, Pichia methanolica, Pichia kluyveri, Yarrowia lipolytica, Candida sp., Candida utilis, Candida cacaoi, Geotrichum sp., and Geotrichum fermentans.
  • the transformation of the yeast cells may for instance be effected by protoplast formation followed by transformation in a manner known perse.
  • the medium used to cultivate the cells may be any conventional medium suitable for growing yeast organisms.
  • the secreted insulin precursor of the invention may be recovered from the medium by conventional procedures including separating the yeast cells from the medium by centrifugation, filtration or catching the insulin by an ion exchange matrix or by a reverse phase absorption matrix, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt, e.g. ammonium sulphate, followed by purification by a variety of chromatographic procedures, e.g. ion exchange chromatography, affinity chromatography, or the like.
  • a salt e.g. ammonium sulphate
  • the insulin precursors of the invention may be expressed with an N-terminal amino acid residue extension, as described in U.S. Patent No. 5,395,922, and European Patent No. 765.395A, both of which patents are herein specifically incorporated by reference.
  • the extension is found to be stably attached to the insulin precursor of the invention during fermentation, protecting the N-terminal end of the insulin precursor against the proteolytic activity of yeast proteases such as DPAP.
  • the presence of an N-terminal extension on the insulin precursor may also serve as a protection of the N-terminal amino group during chemical processing of the protein, i.e. it may serve as a substitute for a BOC (t-butyl- oxycarbonyl) or similar protecting group.
  • the N-terminal extension may be removed from the recovered insulin precursor by means of a proteolytic enzyme which is specific for a basic amino acid (e.g., Lys) so that the terminal extension is cleaved off at the Lys residue.
  • proteolytic enzymes are trypsin or Achromobacter lyticus protease.
  • the insulin precursor of the invention will be subjected to various in vitro procedures to remove the possible N-terminal extension sequence and the mini C-peptide to give desB30 insulin. DesB30 insulin may then be converted into human insulin by adding a Thr in position B30.
  • plasmids which are characterized by containing the Schizosaccharomyces pombe triose phosphate isomerase gene (POT) for the purpose of plasmid selection and stabilization in S. cerevisiae.
  • POT Schizosaccharomyces pombe triose phosphate isomerase gene
  • the plasmids furthermore contain the S. cerevisiae triose phosphate isomerase promoter and terminator.
  • S. cerevisiae triose phosphate isomerase promoter and terminator are similar to the corresponding sequences in plasmid pKFN1003 (described in WO 90/100075) as are all sequences except the sequence of the EcoRI-Xi al fragment encoding the fusion of the leader and insulin precursor.
  • EcoRI-Xfcal fragment of pKFN1003 or a similar C- POT type expreseeion plasmid is simply replaced by an EcoRI-Xfoal fragment encoding the leader insulin precursor of interest.
  • EcoR ⁇ -Xba ⁇ fragments may be synthesized using synthetic oligonucleotides and PCR according to standard techniques.
  • Yeast transformants were prepared by transformation of the host strain: S. cerevisiae strain MT663 (MATa/MAT ⁇ pep4-3/pep4-3 HIS4/his4 tpi::LEU2/tpi::LEU2 Cir + ).
  • the yeast strain MT663 was deposited in the Deutsche Sammlung von Mikroorganismen und Zellkulturen in connection with filing WO 92/11378 and was given the deposit number DSM 6278.
  • MT663 was grown on YPGaL (1% Bacto yeast extract, 2% Bacto peptone, 2% galactose, 1% lactate) to an O.D. at 600 nm of 0.6.
  • the suspension was incubated at 30°C for 15 minutes, centrifuged and the cells resuspended in 10 ml of a solution containing 1.2 M sorbitol, 10 mM Na 2 EDTA, 0.1 M sodium citrate, pH 0 5.8, and 2 mg Novozym®234.

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Abstract

L'invention concerne de nouveaux précurseurs d'insuline comprenant un peptide de connexion (mini-peptide C) contenant jusqu'à 4 résidus d'aminoacides, et comprenant une séquence Glu-Asp, Glu-Glu, Asp-Asp ou Asp-Glu, que l'on peut préparer avec un rendement élevé en micro-organismes transformés, par exemple en levure. On peut convertir les précurseurs en insuline humaine ou en insuline humaine desB30.
PCT/DK2004/000209 2003-03-27 2004-03-26 Procede de fabrication de precurseurs de l'insuline humaine et insuline humaine Ceased WO2004085472A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015015518A3 (fr) * 2013-07-31 2015-04-16 Biogenomics Limited Procédé de production d'insuline et d'analogues d'insuline
US10392429B2 (en) * 2014-10-06 2019-08-27 Case Western Reserve University Biphasic single-chain insulin analogues

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015015518A3 (fr) * 2013-07-31 2015-04-16 Biogenomics Limited Procédé de production d'insuline et d'analogues d'insuline
US10000544B2 (en) 2013-07-31 2018-06-19 Biogenomics Limited Process for production of insulin and insulin analogues
US10392429B2 (en) * 2014-10-06 2019-08-27 Case Western Reserve University Biphasic single-chain insulin analogues
AU2015328222B2 (en) * 2014-10-06 2020-08-20 Case Western Reserve University Biphasic single-chain insulin analogues
AU2015328222B9 (en) * 2014-10-06 2020-12-24 Case Western Reserve University Biphasic single-chain insulin analogues
US11142560B2 (en) 2014-10-06 2021-10-12 Case Western Reserve University Biphasic single-chain insulin analogues

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