DK2330201T3 - Fremgangsmåder til syntese af heteromultimere polypeptider i gær ved anvendelse af en haploid parringsstrategi - Google Patents
Fremgangsmåder til syntese af heteromultimere polypeptider i gær ved anvendelse af en haploid parringsstrategi Download PDFInfo
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- DK2330201T3 DK2330201T3 DK10183188.1T DK10183188T DK2330201T3 DK 2330201 T3 DK2330201 T3 DK 2330201T3 DK 10183188 T DK10183188 T DK 10183188T DK 2330201 T3 DK2330201 T3 DK 2330201T3
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- pichia pastoris
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
- C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
- C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Claims (14)
1. Fremgangsmåde til syntetisering og udvinding fra en Pichia pastoris-gær af et sekreteret, biologisk aktivt, heterologt (ikke-gær) heteromultimert immunoglobulinprotein, der består af mindst to ikke-identiske underenhedspolypeptidkæder, kendetegnet ved følgende trin: (i) fremstilling af en eller flere stabile diploide Pichia pastoris-gærceller ved parring eller sfæroplastfusion af haploide Pichia pastoris-gærceller under betingelser, således at parrings- eller sfæroplastfusionshændelseme resulterer i fremstilling af en eller flere diploide Pichia pastoris-gærceller, hvor den eller de diploide Pichia pastoris- celler efter parringen eller fusionen omfatter ekspressionskonstrukter, der muliggør ekspression og sekretion af de mindst to ikke-identiske polypeptidkæder, der udgør det hele heteromultimere immunoglobulinprotein, og hvilke stabile diploide Pichia pastoris-gærceller er i stand til samles og længerevarende udtrykke og sekretere det hele heteromultimere immunoglobulinprotein i et dyrkningsmedium, der indeholder den eller de stabile diploide Pichia pastoris-celler, når den eller de diploide Pichia pastoris-celler dyrkes under egnende dyrkningsbetingelser; (ii) efter foretagelse af parringen eller sfæroplastfusionen identificering og isolering af den eller dc resulterende diploide Pichia pastoris-celler, der indeholder ekspressionskonstrukter, som muliggør den stabile ekspression og sekretion af de mindst to ikke-identiske polypeptidkæder, der udgør det hele heteromultimere immunoglobulinprotein, når den eller de diploide Pichia pastoris-gærccWcr dyrkes under egnede dyrkningsbetingelser; (iii) dyrkning af den eller de isolerede diploide Pichia pastoris-celler eller deres diploide afkom i et dyrkningsmedium under betingelser, der resulterer i ekspression, samling og sekretion af det biologisk aktive hele heteromultimere immunoglobulinprotein i dyrkningsmediet; og (iv) udvinding af de resulterende heteromultimere protein fra dyrkningsmediet; hvor parringen eller sfæroplastfusionen foretages ved parring eller fusion af en første haploid Pichia pastoris-gærcelle, der indeholder et første ekspressionskonstrukt, hvilket første ekspressionskonstrukt indeholder nukleinsyresekvenser, der koder for ekspressionen af mindst én underenhed af det hele heteromultimere (intakte) immunoglobulinprotein, operativt bundet til en første gærpromoter; og en anden haploid Pichia pastoris-celle, der indeholder et andet ekspressionskonstrukt, hvilket andet ekspressionskonstrukt omfatter nukleinsyresekvenser, der koder for den eller de resterende underenheder af det heteromultimere immunoglobulinprotein, operativt forbundet med en anden gærpromoter.
2. Fremgangsmåde ifølge krav 1, hvor det heteromultimere immunoglobulinprotein er et kimærisk eller humaniseret antistof.
3. Fremgangsmåde ifølge krav 1, hvor de genetiske konstrukter er integreret i genomet af Pichia pas to ris-gære ellerne.
4. Fremgangsmåde ifølge krav 1, hvor de genetiske konstrukter er indeholdt i det extrakromosale (plasmid)element.
5. Fremgangsmåde ifølge krav 1, hvor den første eller den anden promoter er konstitutiv.
6. Fremgangsmåde ifølge krav 1, hvor den første eller den anden promoter er inducerbar.
7. Fremgangsmåde ifølge krav 1, hvor de diploide Pichia /v/.vtor/.v-gærccllcr dyrkes i et produktionsmedium.
8. Fremgangsmåde ifølge krav 7, hvor produktionsmediet er et minimalt medium, der mangler selektive midler, såsom fordannede aminosyrer eller andre komplekse biomolekyler.
9. Fremgangsmåde ifølge krav 1, hvor de diploide Pichia pastoris-gærceller dyrkes til en høj celledensitet.
10. Fremgangsmåde ifølge krav 9, hvor den høje celledensitet er mindst 50, 100, 300, 400 eller 500 g/L.
11. Fremgangsmåde ifølge krav 1, hvor de diploide Pichia pastoris-gærceller dyrkes under betingelser, der resulterer i niveauer af det biologisk aktive heteromultimere immunoglobulinprotein, som er mindst 50, 100, 500 eller 1000 mg/L.
12. Fremgangsmåde ifølge krav 1, hvor de diploide Pichia pastoris-gærccUcr dyrkes til mindst 20, 50 eller 100 fordoblinger og bevarer høje niveauer af ekspression af det heteromultimere immunoglobulinprotein efter de mindst 20, 50 eller 100 fordoblinger, og hvor efter de mindst 20, 50 eller 100 fordoblinger mindst 99 % af de diploide Pichia pastoris-celler omfatter ekspressionskonstrukter, der muliggør ekspressionen af de kæder, der danner det heteromultimere immunoglobulinprotein.
13. Fremgangsmåde ifølge krav 1, hvor de diploide Pichia pasloris-gærccWcr dyrkes til mindst 20, 50 eller 100 fordoblinger og efter de mindst 20, 50 eller 100 fordoblinger udtrykker det heteromultimere immunoglobulinprotein ved et ekspressions niveau, der højest er reduceret med 20 %, 10 % eller 5 % i forhold til ekspressionens udgangsniveau.
14. Dyrkningsmedium, der indeholder en stabil diploid Pichia pastoris-gærkultur ifølge krav 1, hvor dyrkningsmediet omfatter ekspressionsniveauer af det biologisk aktive heteromultimere immunoglobulinprotein, der er mindst ca. 50 mg/liter, 100 mg/liter, 500 mg/liter eller 1000 mg/liter eller mere, og hvor celledensiteten af de diploide Pichia pastoris-CQhQr i kulturen er mindst ca. 50 g/L, 100 g/L, 300 g/L, 400 g/L, 500 g/L eller mere.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US51387603P | 2003-10-22 | 2003-10-22 | |
| EP04796313A EP1678314B1 (en) | 2003-10-22 | 2004-10-22 | Methods of synthesizing heteromultimeric polypeptides in yeast using a haploid mating strategy |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| DK2330201T3 true DK2330201T3 (da) | 2017-07-24 |
Family
ID=34520146
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| DK10183188.1T DK2330201T3 (da) | 2003-10-22 | 2004-10-22 | Fremgangsmåder til syntese af heteromultimere polypeptider i gær ved anvendelse af en haploid parringsstrategi |
| DK04796313.7T DK1678314T3 (da) | 2003-10-22 | 2004-10-22 | Fremgangsmåde til syntetisering af heteromultimere polypeptider i gær ved anvendelse af en haploid parringsstrategi |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| DK04796313.7T DK1678314T3 (da) | 2003-10-22 | 2004-10-22 | Fremgangsmåde til syntetisering af heteromultimere polypeptider i gær ved anvendelse af en haploid parringsstrategi |
Country Status (17)
| Country | Link |
|---|---|
| US (7) | US8268582B2 (da) |
| EP (2) | EP1678314B1 (da) |
| JP (3) | JP4994038B2 (da) |
| CN (2) | CN1871359B (da) |
| AU (2) | AU2004283299B2 (da) |
| CA (1) | CA2541651C (da) |
| DK (2) | DK2330201T3 (da) |
| ES (2) | ES2630224T3 (da) |
| HK (1) | HK1093222A1 (da) |
| HU (1) | HUE033349T2 (da) |
| IL (2) | IL174783A (da) |
| NO (1) | NO20062134L (da) |
| NZ (1) | NZ547279A (da) |
| PL (1) | PL1678314T3 (da) |
| PT (1) | PT1678314E (da) |
| SI (1) | SI1678314T1 (da) |
| WO (1) | WO2005040395A1 (da) |
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