DK2115126T3 - Anvendelse af kobber og glutamat i cellekulturer til fremstilling af polypeptider - Google Patents

Anvendelse af kobber og glutamat i cellekulturer til fremstilling af polypeptider Download PDF

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DK2115126T3
DK2115126T3 DK08731084T DK08731084T DK2115126T3 DK 2115126 T3 DK2115126 T3 DK 2115126T3 DK 08731084 T DK08731084 T DK 08731084T DK 08731084 T DK08731084 T DK 08731084T DK 2115126 T3 DK2115126 T3 DK 2115126T3
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polypeptide
cell culture
cell
cells
temperature range
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DK08731084T
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Denis Drapeau
Gregory Hiller
Jessica Snow
Yen-Tung Luan
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Wyeth Llc
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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
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0018—Culture media for cell or tissue culture
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/20—Transition metals
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/32—Amino acids
    • 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
    • C12N2510/00—Genetically modified cells
    • C12N2510/02—Cells for production

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
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  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
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  • General Engineering & Computer Science (AREA)
  • Cell Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)

Claims (29)

1. Fremgangsmåde til fremstilling af et polypeptid i en cellekultur omfattende de følgende trin: at dyrke pattedyrceller, som indeholder et gen, der koder et polypeptid af interesse, i et cellekulturmedium omfattende mellem 0,5 og 5 μΜ kobber og mellem 1,7 og 33 mM glutamat; at fastholde kulturen i et første temperaturområde i en første tidsperiode, der er tilstrækkelig til at lade cellerne reproducere sig til en levedygtig celletæt-hed inden for et område på 20 % - 80 % af den maksimalt mulige levedygtige celletæthed, hvis kulturen blev fastholdt i det første temperaturområde; at skifte kulturen til et andet temperaturområde, hvor mindst en temperatur fra det andet temperaturområde er lavere end den laveste temperatur fra det første temperaturområde; at fastholde kulturen i en anden tidsperiode under betingelser og i et tidsrum, der er tilstrækkeligt til at tillade ekspression af polypeptidet, hvor fraktionen af misudfoldet og/eller aggregeret polypeptid, i forhold til den totale mængde fremstillet polypeptid, reduceres sammenlignet med fraktionen af misudfoldet og/eller aggregeret polypeptid, som ville blive observeret under i øvrigt identiske betingelser i et i øvrigt identisk medium, som mangler kobber og glutamat; hvor polypeptidet er TNFR- Ig.
2. Fremgangsmåde ifølge krav 1, hvor det første temperaturområde omfatter et temperaturområde, som er 30 til 42 grader Celsius.
3. Fremgangsmåde ifølge krav 1, hvor det andet temperaturområde omfatter et temperaturområde, som er 25 til 41 grader Celsius.
4. Fremgangsmåde ifølge krav 1, omfattende et andet skiftetrin efter det første skiftetrin omfattende at skifte kulturen til et tredje temperaturområde, hvor mindst en temperatur fra det tredje temperaturområde er lavere end den laveste temperatur fra det andet temperaturområde.
5. Fremgangsmåde ifølge krav 4, hvor det tredje temperaturområde omfatter et temperaturområde, som er 25 til 40 grader Celsius.
6. Fremgangsmåde ifølge krav 1, hvor glutamin-startkoncentrationen af cellekulturmediet er mindre end eller lig med 4 mM.
7. Fremgangsmåde ifølge krav 1, hvor cellekulturen yderligere suppleres med 2 gram pr. liter glucose.
8. Fremgangsmåde ifølge krav 1, hvor cellekulturen yderligere suppleres med supplerende bestanddele.
9. Fremgangsmåde ifølge krav 8, hvor de supplerende bestanddele forefindes i et fodermedium.
10. Fremgangsmåde ifølge krav 9, hvor de supplerende bestanddele forefindes i flere intervaller.
11. Fremgangsmåde ifølge et eller flere af de foregående krav, hvor cellekulturmediet er defineret.
12. Fremgangsmåde ifølge krav 11, hvor det definerede cellekulturmedium ikke indeholder tilføjet serum eller hydrolysater.
13. Fremgangsmåde ifølge krav 11, hvor det definerede cellekulturmedium er proteinfrit.
14. Fremgangsmåde ifølge et af de foregående krav, hvor cellekulturmediet omfatter mellem 0,7 og 1,5 μΜ kobber.
15. Fremgangsmåde ifølge krav 14, hvor cellekulturmediet omfatter 1 μΜ kobber.
16. Fremgangsmåde ifølge et af de foregående krav, hvor cellekulturmediet omfatter mellem 3 og 7 pM glutamat.
17. Fremgangsmåde ifølge krav 16, hvor cellekulturmediet omfatter 5 μΜ glutamat.
18. Fremgangsmåde til fremstilling af et polypeptid i en cellekultur omfattende de følgende trin: at dyrke pattedyrceller, som indeholder et gen, der koder et polypeptid af interesse, i et cellekulturmedium omfattende mellem 0,5 og 5 μΜ kobber og mellem 1,7 og 33 mM glutamat; at fastholde kulturen i et første temperaturområde i en første tidsperiode, der er tilstrækkelig til at lade cellerne reproducere sig til en levedygtig celletæt-hed inden for et område på 20 % - 80 % af den maksimalt mulige levedygtige celletæthed, hvis kulturen blev fastholdt i det første temperaturområde; at skifte kulturen til et andet temperaturområde, hvor mindst en temperatur fra det andet temperaturområde er lavere end den laveste temperatur fra det første temperaturområde; at fastholde kulturen i en anden tidsperiode under betingelser og i et tidsrum, der er tilstrækkeligt til at tillade ekspression af polypeptidet, hvor glycosyle-ringsmønsteret af det eksprimerede polypeptid øges i forhold til det glycosy-leringsmønster, der ville blive observeret på det eksprimerede polypeptid under i øvrigt identiske betingelser i et i øvrigt identisk medium, som mangler kobber og glutamat; hvor polypeptidet er TNFR- Ig.
19. Fremgangsmåde ifølge krav 18, hvor det øgede glycosyleringsmønster for det eksprimerede polypeptid omfatter en øgning i total sialylation.
20. Fremgangsmåde ifølge krav 18, hvor cellekulturmediet er defineret.
21. Fremgangsmåde ifølge krav 20, hvor det definerede cellekulturmedium ikke indeholder tilføjet serum eller hydrolysater.
22. Fremgangsmåde ifølge krav 20, hvor det definerede cellekulturmedium er proteinfrit.
23. Fremgangsmåde ifølge et af kravene 18-22, hvor cellekulturmediet omfatter mellem 0,7 og 1,5 μΜ kobber.
24. Fremgangsmåde ifølge krav 23, hvor cellekulturmediet omfatter 1 μΜ kobber.
25. Fremgangsmåde ifølge et af kravene 18-22, hvor cellekulturmediet omfatter mellem 3 og 7 μΜ glutamat.
26. Fremgangsmåde ifølge krav 25, hvor cellekulturmediet omfatter 5 μΜ glutamat.
27. Fremgangsmåde ifølge et af kravene 1-26, hvor polypeptidet er TNFR-Fc.
28. Fremgangsmåde ifølge et af kravene 1-27, hvor dyrkningstrinnet omfatter at dyrke pattedyrcellerne i en batchkultur.
29. Fremgangsmåde ifølge et af kravene 1-28, hvor polypeptidet eller proteinet isoleres og/eller oprenses.
DK08731084T 2007-03-02 2008-02-29 Anvendelse af kobber og glutamat i cellekulturer til fremstilling af polypeptider DK2115126T3 (da)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US89274907P 2007-03-02 2007-03-02
PCT/US2008/055447 WO2008109410A1 (en) 2007-03-02 2008-02-29 Use of copper and glutamate in cell culture for production of polypeptides

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US (1) US9012180B2 (da)
EP (2) EP2924113B1 (da)
JP (1) JP5586235B2 (da)
KR (1) KR20090127326A (da)
CN (1) CN101679941A (da)
AU (1) AU2008223133A1 (da)
CA (1) CA2679941C (da)
DK (1) DK2115126T3 (da)
ES (2) ES2728912T3 (da)
HU (1) HUE026403T2 (da)
IL (1) IL200622A0 (da)
MX (1) MX2009009240A (da)
PL (1) PL2115126T3 (da)
PT (1) PT2115126E (da)
RU (1) RU2009132986A (da)
SI (1) SI2115126T1 (da)
WO (1) WO2008109410A1 (da)
ZA (1) ZA200906061B (da)

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MX2009009240A (es) 2009-09-08
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EP2924113B1 (en) 2019-04-10
EP2924113A1 (en) 2015-09-30
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HUE026403T2 (en) 2016-06-28
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EP2115126A1 (en) 2009-11-11
CA2679941A1 (en) 2008-09-12
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