DK2356247T3 - Fremgangsmåde til fremstilling af serum-frit insulin-frit faktor vii. - Google Patents
Fremgangsmåde til fremstilling af serum-frit insulin-frit faktor vii. Download PDFInfo
- Publication number
- DK2356247T3 DK2356247T3 DK09826577.0T DK09826577T DK2356247T3 DK 2356247 T3 DK2356247 T3 DK 2356247T3 DK 09826577 T DK09826577 T DK 09826577T DK 2356247 T3 DK2356247 T3 DK 2356247T3
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- Prior art keywords
- insulin
- free
- serum
- culture
- cells
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Claims (30)
1. Fremgangsmåde til fremstilling af rekombinant human Faktor VII (FVII) omfattende at: a) tilvejebringe en kinesisk hamster-ovarie (CFIO)-cellelinie (ENG: Chinese Flam-ster Ovary cell line), som udtrykker rekombinant human FVII; og b) dyrke CFIO-cellen i serum-frit medie, som er uden insulin, hvor CFIO-cellen fremstilles ved at tilpasse en CFIO-cellelinie, som udtrykker rekombinant human FVII modificeret til vækst i serum-fri kultur til at vokse i cellekultur uden insulin ved serielt at dyrke CFIO-cellelinien i aftagende mængder af insulin for at tilvejebringe en FVII-fremstillende CFIO-cellelinie, som vokser uden insulin, og hvor den serielle dyrkning omfatter at pulse på hinanden følgende passager af CFIO-celler med aftagende koncentrationer af insulin.
2. Fremgangsmåde ifølge krav 1, hvor CFIO-cellelinien, som udtrykker rekombinant human FVII modificeret til vækst i serum-fri kultur er cellelinien lE9-cellelinie deponeret under tilgangsnummeret 08100801 ved The European Collection of Cell Cultures, Porton Down, Storbritanien, den 8. oktober, 2008.
3. Fremgangsmåde ifølge krav 1, hvor cellekulturmediumet omfatter vitamin Kl.
4. Fremgangsmåde ifølge krav 1, hvor et trin i pulseringen omfatter at dyrke en CFIO-cellelinie op til dag 14 i standard serum-frit medie, som indeholder insulin, og på dag 14 at overføre celle-linien til et serum-frit, insulin-frit medie.
5. Fremgangsmåde ifølge krav 4, omfattende derefter at dyrke cellerne i et serum-frit, insulin-frit medie i 1 til 3 dage og derefter at overføre cellerne til et insulin-holdigt medie, som omfatter 0,2 mg/ml insulin og at dyrke CFIO-cellelinierne i det insulin-holdige medie i 1 til 3 dage, og efterfølgende, efter vækst i 1 til 3 dage i insulin-holdigt medie at overføre CFIO-cellelinien til serum-frit, insulin-frit medie
6. Fremgangsmåde ifølge krav 5, omfattende derefter at dyrke cellerne i et serum-frit, insulin-frit medie i fortrinsvis 1 dag og derefter at overføre cellerne til et insulin-holdigt medie, som omfatter 0,070 mg/ml insulin og at dyrke CFIO-cellelinien i det insulin-holdige medie i 3 til 8 dage, og, efter vækst i 3 til 8 dage i insulin-holdigt medie, at overføre CFIO-cellelinien til serum-frit, insulin-frit medie.
7. Fremgangsmåde ifølge krav 6, omfattende derefter at dyrke cellerne i et serum-frit, insulin-frit medie i 1 dag og derefter at overføre cellerne til et insulin-holdigt medie, som omfatter 0,030 mg/ml insulin og at dyrke CFIO-cellelinien i det insulin-holdige medie i 3 til 9 dage, og efter vækst i 3 til 9 dage i insulin-holdigt medie, at overføre CFIO-cellelinien til serum-frit, insulin-frit medie.
8. Fremgangsmåde ifølge krav 7, omfattende derefter at dyrke cellerne i et serum-frit, insulin-frit medie i 1 dag og derefter at overføre cellerne til et insulin-holdigt medie, som omfatter 0,016 mg/ml insulin og at dyrke CHO-cellelinien i det insulin-holdige medie i 3 til 9 dage, og derefter at overføre CHO-cellelinien til serum-frit, insulin-frit medie.
9. Fremgangsmåde til tilpasning af en FVII-fremstillende cellelinie, som vokser i serum-frit medie til en cellelinie, som vokser i serum-frit, insulin-frit medie, omfattende at pulse på hinanden følgende passager af CHO-celler med aftagende koncentrationer af insulin, hvor pulseringen omfatter a) at dyrke cellelinien i 1 til 9 dage i insulin-holdigt medie efterfulgt af b) vækst i insulin-frit, serum-frit medie i 1 til 3 dage og at gentage trin a) og b), hvor mediet i hvert på hinanden følgende trin a) indeholder omtrent halvdelen af koncentrationen af insulin som i det foregående trin a) indtil mediet indeholder intet detekter-bart insulin.
10. Fremgangsmåde ifølge krav 9, hvor fremgangsmåden omfatter at: a) dyrke en CHO-cellelinie op til dag 14 i standard serum-frit medie, som indeholder insulin og på dag 14 at overføre cellelinien til et serum-frit, insulin-frit medie; b) dyrke cellerne i trin b) i et serum-frit, insulin-frit medie i 1 til 2 dage og derefter at overføre cellerne til et insulin-holdigt medie, som omfatter 0,2 mg/ml insulin og at dyrke CHO-cellelinien i det insulin-holdige medie i 1 til 3 dage; c) efter vækst i 1 til 3 dage i insulin-holdigt medie af trin b), at overføre CHO-cellelinien til serum-frit, insulin-frit medie; d) dyrke cellerne i trin c) i et serum-frit, insulin-frit medie i 1 til 3 dage og derefter at overføre cellerne til et insulin-holdigt medie, som omfatter 0,070 mg/ml insulin og at dyrke CHO-cellelinien i det insulin-holdige medie i 3 til 8 dage; e) efter vækst i 3 til 8 dage i insulin-holdigt medie i trin d), at overføre CHO-cellelinien til serum-frit, insulin-frit medie; f) dyrke cellerne i trin e) i et serum-frit, insulin-frit medie i 1 til 2 dage og derefter at overføre cellerne til et insulin-holdigt medie, som omfatter 0,030 mg/ml insulin og at dyrke CHO-cellelinien i det insulin-holdige medie i 3 til 9 dage; g) efter vækst i 3 til 9 dage i insulin-holdigt medie i trin f), at overføre CHO-cellelinien til serum-frit, insulin-frit medie; h) dyrke cellerne i trin g) i et serum-frit, insulin-frit medie i 1 til 2 dage og derefter at overføre cellerne til et insulin-holdigt medie, som omfatter 0,016 mg/ml insulin og at dyrke CHO-cellelinien i det insulin-holdige medie i 3 til 9 dage; og i) efter vækst i 3 til 9 dage i insulin-holdigt medie i trin h), at overføre CHO-cellelinien til serum-frit, insulin-frit medie, hvor cellerne i trin i) er tilpasset til langsigtet vækst i serum-frit, insulin-frit medie.
11. CHO-celle, der kan tilvejebringes ved fremgangsmåden ifølge krav 9 eller krav 10.
12. CHO-cellen ifølge krav 11, hvor CHO-cellen kan tilvejebringes ved fremgangsmåden ifølge krav 9, og hvor CHO-cellen er en rekombinant CHO-celle, som udtrykker human FVII og er tilpasset til vækst i et insulin-frit medie, som er uden dyre-afledte bestanddele.
13. Fremgangsmåde til storskala fremstilling af en FVII eller et FVII-relateret po-lypeptid i CHO-celler, hvor fremgangsmåde omfatter at: (a) inokulere en CHO-celle ifølge krav 11 i en dyrkningsbeholder indeholdende serum-frit, insulin-frit medium og at opformere den mammale cellekultur mindst indtil cellerne opnår en forudbestemt massefylde; (b) overføre det opformerede stamkultur til en storskala dyrkningsbeholder indeholdende serum-frit insulin-frit medium; (c) opformere den storskala dyrkningsbeholder i serum-frit insulin-frit medium, mindst indtil cellerne opnår en forudbestemt vægtfylde; (d) opretholde kulturen tilvejebragt i trin (c) i serum-frit insulin-frit medium, under betingelser hensigtsmæssig til FVII-ekspression eller FVII-relateret polypeptidekspres-sion; og (e) indvinde FVII'en eller det FVII-relaterede polypeptid fra den opretholdte kultur.
14. Fremgangsmåde ifølge krav 13, yderligere omfattende, forud for trin (b), at gentage trin (a) ved anvendelse af stamkulturbeholdere af progressive stigende dimensioner.
15. Fremgangsmåde ifølge krav 13, yderligere omfattende at: opretholde kulturen tilvejebragt i trin (c) i serum-frit, insulin-frit medie ved regelmæssig høstning af dyrkningsmediet og udskiftning med friskt medium.
16. Fremgangsmåde ifølge krav 13, hvor fremgangsmåden er valgt fra gruppen bestående af: A) mikrobærereproces; B) en suspensionsproces; og C) en kemostatdyrkningsproces.
17. Fremgangsmåde ifølge krav 16, hvor fremgangsmåden er en mikrobærerpro-ces, og hvor fremgangsmåden er en makroporøs bærestofproces.
18. Fremgangsmåde ifølge krav 16, hvor fremgangsmåden er en mikrobærerpro-ces, og hvor fremgangsmåden er valgt fra gruppen bestående af: A) en standard mikrobærerproces; og B) en mikrobærerperfusionsproces.
19. Fremgangsmåde ifølge krav 16, hvor fremgangsmåden er en suspensionsproces, og hvor fremgangsmåden er valgt fra gruppen bestående af: A) en perfusionsproces; og B) en batch-/udtræknings-påfyldnings-proces (ENG: draw-fill process).
20. Fremgangsmåde ifølge krav 19, hvor fremgangsmåden er en batch-/udtræknings-påfyldnings-proces (ENG: draw-fill process), og hvor fremgangsmåden er valgt fra gruppen bestående af: A) en simpel batchproces; B) en fed-batch-proces; og C) en udtræknings-påfyldnings-proces (ENG: draw-fill process).
21. Fremgangsmåde ifølge krav 13, hvor cellerne, forud for inokuleringstrinnet, er i stand til at vokse i suspensionskultur.
22. Fremgangsmåde ifølge krav 13, hvor den ønskede FVII eller det FVII-relaterede polypeptid er human FVII eller et humant FVII-relateret polypeptid.
23. Fremgangsmåde ifølge krav 13, hvor FVII eller et FVII-relateret polypeptid fremstilles ved et niveau omkring mindst 1 mg/l/dag af kultur; fortrinsvis, hvor FVII eller et FVII-relateret polypeptid fremstilles ved et niveau omkring mindst 2,5 mg/l/dag af kultur.
24. Fremgangsmåde ifølge krav 23, hvor FVII eller et FVII-relateret polypeptid fremstilles ved et niveau omkring mindst 5 mg/l/dag af kultur.
25. Fremgangsmåde ifølge krav 24, hvor FVII eller et FVII-relateret polypeptid fremstilles ved et niveau omkring mindst 8 mg/l/dag af kultur.
26. Fremgangsmåde ifølge krav 24, hvor FVII eller et FVII-relateret polypeptid fremstilles ved et niveau af omkring 3-4 mg/l/dag.
27. Fremgangsmåde ifølge krav 24, hvor enhederne af FVII eller af FVII-relaterede polypeptider fremstilles i mindst 5000 U/l/dag, fortrinsvis, hvor enhederne af FVII eller FVII-relaterede polypeptid fremstilles i mindst 7000 U/l/dag, mere fortrinsvis, hvor aktivitetsenhederne af FVII eller af et FVII-relateret polypeptid fremstilles i omkring 7000-10,000 U/l/dag.
28. Fremgangsmåde ifølge krav 13, yderligere omfattende at: opretholde kulturen tilvejebragt i trin (c) i dyreprodukt-frit, insulin-frit medie ved regelmæssig at høste en del af supernantantkulturen efter sedimentering af de celleindeholdende bærestoffer og erstatning med friskt medium.
29. Fremgangsmåde ifølge krav 28, yderligere omfattende at: afkøle kulturen til en forudbestemt temperatur under setpunktet af dyrkningen før sedimenteringen af bærestoffer, fortrinsvis hvor kulturen er kølet til en temperatur fra 5°C til 30°C under set-punktstemperaturen af dyrkningen før sedimenteringen af bærestoffer, fortrinsvis hvor kulturen er kølet til en temperatur fra 5°C til 20°C under setpunktstemperaturen af dyrkningen, fortrinsvis hvor kulturen er kølet til en temperatur fra 5°C to 15°C under setpunktstemperaturen af dyrkningen, fortrinsvis hvor kulturen er kølet til en temperatur fra omkring 10°C under setpunktstemperaturen af dyrkningen.
30. En fremgangsmåde ifølge krav 1, krav 9 eller krav 13, hvor mediet er et serum-frit DMEM/FIAM'er F12-baseret formulering suppleret med én eller flere af de følgende tilsætningsstoffer i) glutamin; slutkoncentration 0,9 g/l, ii) jernsulfat x7FI20 0,0006 g/l, iv) putrescin, x 2HCI 0,0036g/l, vi) vitamin Kl 0,0025 g/l, vii) Synperonic; slutkoncentration 1 g/l, phenolrød 0,008 g/l, ethanolamin 0,00153g/l, og Na-hydrogencarbonat 2g/l).
Applications Claiming Priority (2)
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| US11379208P | 2008-11-12 | 2008-11-12 | |
| PCT/US2009/063338 WO2010056584A1 (en) | 2008-11-12 | 2009-11-05 | Method of producing serum-free insulin-free factor vii |
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| DK2356247T3 true DK2356247T3 (da) | 2015-09-21 |
| DK2356247T4 DK2356247T4 (da) | 2019-06-03 |
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| US (1) | US20100120093A1 (da) |
| EP (2) | EP2356247B2 (da) |
| JP (1) | JP5719301B2 (da) |
| AU (1) | AU2009314345B2 (da) |
| CA (1) | CA2742107A1 (da) |
| DK (1) | DK2356247T4 (da) |
| ES (1) | ES2547881T5 (da) |
| HK (1) | HK1219113A1 (da) |
| HR (1) | HRP20151054T4 (da) |
| HU (1) | HUE026210T2 (da) |
| IL (1) | IL212355A (da) |
| PL (1) | PL2356247T5 (da) |
| PT (1) | PT2356247E (da) |
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| JP2013535981A (ja) * | 2010-08-20 | 2013-09-19 | ワイス・エルエルシー | 成長因子不含適合細胞の細胞培養 |
| US20120076779A1 (en) | 2010-09-17 | 2012-03-29 | Baxter Healthcare S.A. | STABILIZATION OF IMMUNOGLOBULINS AND OTHER PROTEINS THROUGH AQUEOUS FORMULATIONS WITH SODIUM CHLORIDE AT WEAK ACIDIC TO NEUTRAL ph |
| US9217168B2 (en) | 2013-03-14 | 2015-12-22 | Momenta Pharmaceuticals, Inc. | Methods of cell culture |
| AR095196A1 (es) | 2013-03-15 | 2015-09-30 | Regeneron Pharma | Medio de cultivo celular libre de suero |
| KR20150026024A (ko) * | 2013-08-30 | 2015-03-11 | 한미약품 주식회사 | 인간 혈액응고 7인자 유도체의 대량 생산 방법 |
| US11286292B2 (en) | 2014-02-07 | 2022-03-29 | Cmc Biologics A/S | Bioreactor arrangement and continuous process for producing and capturing a biopolymer |
| NZ724826A (en) * | 2014-04-16 | 2022-07-29 | Cmc Biologics As | A high cell density fill and draw fermentation process |
| AU2015269365A1 (en) | 2014-06-04 | 2016-12-15 | Amgen Inc. | Methods for harvesting mammalian cell cultures |
| US12297451B1 (en) | 2019-10-25 | 2025-05-13 | Regeneron Pharmaceuticals, Inc. | Cell culture medium |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US4456591A (en) | 1981-06-25 | 1984-06-26 | Baxter Travenol Laboratories, Inc. | Therapeutic method for activating factor VII |
| GR860984B (en) | 1985-04-17 | 1986-08-18 | Zymogenetics Inc | Expression of factor vii and ix activities in mammalian cells |
| GB9022545D0 (en) * | 1990-10-17 | 1990-11-28 | Wellcome Found | Culture medium |
| US5997864A (en) | 1995-06-07 | 1999-12-07 | Novo Nordisk A/S | Modified factor VII |
| US5804420A (en) * | 1997-04-18 | 1998-09-08 | Bayer Corporation | Preparation of recombinant Factor VIII in a protein free medium |
| AT407255B (de) * | 1997-06-20 | 2001-02-26 | Immuno Ag | Rekombinanter zellklon mit erhöhter stabilität in serum- und proteinfreiem medium und verfahren zur gewinnung des stabilen zellklons |
| AT409379B (de) † | 1999-06-02 | 2002-07-25 | Baxter Ag | Medium zur protein- und serumfreien kultivierung von zellen |
| DK1200561T3 (da) † | 1999-08-05 | 2006-10-16 | Baxter Ag | Rekombinant stabil celleklon, dens fremstilling og anvendelse |
| US20030204338A1 (en) | 2002-04-22 | 2003-10-30 | Peter Martinek | Method and measurement probe for the performance of measurements in water supply systems |
| US20040185535A1 (en) † | 2003-03-21 | 2004-09-23 | Giles Wilson | Industrial-scale serum-free production of recombinant FVII in mammalian cells |
| BR0114373A (pt) * | 2000-10-02 | 2004-02-17 | Novo Nordisk As | Célula hospedeira eucariótica, métodos para a produção de fvii e de fviia ou de suas variantes, de uma proteìna dependente de vitamina k e de uma célula hospedeira eucariótica produtora de fvii ou de seu análogo, vetor recombinante, fvii e fviia recombinante ou suas variantes, e, proteìna dependente de vitamina k recombinante |
| EP1434857B1 (en) * | 2001-10-02 | 2007-08-01 | Novo Nordisk Health Care AG | Method for production of recombinant proteins in eukaryote cells |
| US20050214289A1 (en) * | 2004-03-26 | 2005-09-29 | Human Genome Sciences, Inc. | Antibodies against Nogo receptor |
| US20060094104A1 (en) | 2004-10-29 | 2006-05-04 | Leopold Grillberger | Animal protein-free media for cultivation of cells |
| EP1851305B1 (en) * | 2005-02-11 | 2012-01-18 | Novo Nordisk Health Care AG | Production of a polypeptide in a serum-free cell culture liquid containing plant protein hydrolysate |
| CN101300342A (zh) † | 2005-11-02 | 2008-11-05 | 惠氏公司 | 用于使哺乳动物细胞具备适应性的方法 |
| ES2474573T3 (es) † | 2006-01-04 | 2014-07-09 | Baxter International Inc | Medio de cultivo celular sin oligop�ptidos |
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Also Published As
| Publication number | Publication date |
|---|---|
| PL2356247T5 (pl) | 2019-10-31 |
| SI2356247T1 (sl) | 2015-11-30 |
| HK1219113A1 (en) | 2017-03-24 |
| PT2356247E (pt) | 2015-10-26 |
| CA2742107A1 (en) | 2010-05-20 |
| EP2977461A1 (en) | 2016-01-27 |
| JP2012508562A (ja) | 2012-04-12 |
| IL212355A0 (en) | 2011-06-30 |
| US20100120093A1 (en) | 2010-05-13 |
| JP5719301B2 (ja) | 2015-05-13 |
| ES2547881T5 (es) | 2019-10-16 |
| AU2009314345B2 (en) | 2015-02-12 |
| EP2356247A4 (en) | 2013-01-02 |
| PL2356247T3 (pl) | 2016-04-29 |
| WO2010056584A1 (en) | 2010-05-20 |
| SI2356247T2 (sl) | 2019-08-30 |
| EP2356247A1 (en) | 2011-08-17 |
| IL212355A (en) | 2016-04-21 |
| HRP20151054T4 (hr) | 2019-11-15 |
| EP2356247B2 (en) | 2019-05-15 |
| EP2356247B1 (en) | 2015-07-15 |
| HRP20151054T1 (xx) | 2015-11-20 |
| HUE026210T2 (en) | 2016-05-30 |
| ES2547881T3 (es) | 2015-10-09 |
| AU2009314345A1 (en) | 2011-06-30 |
| DK2356247T4 (da) | 2019-06-03 |
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