ES2613269T3 - Métodos para la producción de glucoproteínas en cultivos de células de mamífero usando glucocorticoides - Google Patents
Métodos para la producción de glucoproteínas en cultivos de células de mamífero usando glucocorticoides Download PDFInfo
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Abstract
Un proceso de cultivo celular para la produccion de una molecula de CTLA4 soluble, que comprende: a) cultivar celulas CHO que producen una molecula de CTLA4 soluble en cultivo celular en condiciones que permiten la produccion de proteinas; y b) anadir niveles no toxicos de glucocorticoide.
Description
proceso de cultivo reivindicado, tales como CHO-K1 (ATCC CCL-61), DG44 (Chasin et al., 1986, Som. Cell Molec. Genet., 12:555-556; y Kolkekar et al., 1997, Biochemistry, 36:10901-10909), línea celular Tet-On CHO-K1 (Clontech), CHO designada ECACC 85050302 (CAMR, Salisbury, Wiltshire, Reino Unido), CHO clon 13 (GEIMG, Genova, Italia), CHO clon B (GEIMG, Genova, Italia), CHO-K1/SF designada ECACC 93061607 (CAMR, Salisbury, Wiltshire, Reino
5 Unido), RR-CHOK1 designada ECACC 92052129 (CAMR, Salisbury, Wiltshire, Reino Unido), células CHO negativas en dihidrofolato reductasa (CHO/-DHFR, Urlaub y Chasin, 1980, Proc. Natl. Acad. Sci. USA, 77:4216) y células dp12.CHO (documento de patente de Estados Unidos n.º 5.721.121). Los ejemplos de dichas células hospedadoras incluyen también células que no están abarcadas por las células CHO cultivadas en el proceso de cultivo celular reivindicado, por ejemplo, células CV1 renales de mono transformadas por SV40 (células COS, COS-7, ATCC CRL1651); células renales embrionarias humanas (por ejemplo, células 293 o células 293 subclonadas para crecimiento en cultivo en suspensión, Graham et al., 1977, J. Gen. Virol., 36:59); células renales de cría de hámster (BHK, ATCC CCL-10); células renales de mono (CV1, ATCC CCL-70); células renales de mono verde africano (VERO-76, ATCC CRL-1587; VERO, ATCC CCL-81); células de Sertoli de ratón (TM4, Mather, 1980, Biol. Reprod., 23:243-251); células de carcinoma cervical humano (HELA, ATCC CCL-2); células renales caninas (MDCK, ATCC CCL-34);
15 células pulmonares humanas (W138, ATCC CCL-75); células de hepatoma humano (HEP-G2, HB 8065); células de tumor mamario de ratón (MMT 060562, ATCC CCL-51); células hepáticas de rata búfalo (BRL 3A, ATCC CRL-1442); células TRI (Mather, 1982, Annals NY Acad. Sci., 383:44-68); células MCR 5; células FS4. Las células CHO se cultivan en el proceso de cultivo reivindicado, particularmente, las células CHO/-DHFR.
Las células adecuadas para cultivar en los procedimientos y procesos de la presente invención pueden contener vectores de expresión (constructos) introducidos, por ejemplo, por transformación, transfección, infección o inyección, tales como plásmidos y similares, que alojan secuencias de codificación, o porciones de las mismas, que codifican las proteínas para su expresión y producción en los procedimientos de cultivo. Tales vectores de expresión contienen los elementos necesarios para la transcripción y traducción de la secuencia de codificación insertada.
25 Pueden usarse procedimientos que son bien conocidos y llevados a la práctica por los expertos en la técnica para construir vectores de expresión que contienen secuencias que codifican las proteínas y los polipéptidos producidos, así como los elementos de control transcripcional y transduccional apropiados. Estos procedimientos incluyen técnicas de ADN recombinante in vitro, técnicas sintéticas y recombinación genética in vivo. Tales técnicas están descritas en J. Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview,
- N.Y.
- (EEUU) y en F.M. Ausubel et al., 1989, Current Protocols in Molecular Biology, John Wiley & Sons, Nueva York,
- N.Y.
- (EEUU).
Elementos de control o secuencias reguladoras son las regiones no traducidas del vector, por ejemplo, potenciadores, promotores, regiones no traducidas 5’ y 3’, que interactúan con proteínas celulares huésped para
35 llevar a cabo la transcripción y la traducción. Dichos elementos pueden variar en su fuerza y su especificidad. En función del sistema vectorial y de la célula hospedadora que se utiliza, puede usarse cualquier número de elementos de transcripción y de traducción adecuados, incluidos promotores constitutivos e inducibles. En sistemas celulares de mamíferos, son preferentes los promotores de genes de mamífero o de virus de mamíferos. Los constructos para usar en sistemas de expresión proteica se diseñan para contener al menos un promotor, una secuencia potenciadora (opcionalmente, para sistemas de expresión de mamíferos) y otras secuencias tal como se necesitan o se desean para una transcripción y regulación adecuada de la expresión génica (por ejemplo, secuencias de iniciación y terminación transcripcional, origen de sitios de replicación, secuencias de poliadenilación, por ejemplo, la secuencia poli A de la hormona del crecimiento bovina (BGH)).
45 Como se apreciará por los expertos en la técnica, la selección del vector apropiado, por ejemplo, un plásmido, componentes para una transcripción, expresión y aislamiento apropiados de proteínas producidas en sistemas de expresión eucariótico (por ejemplo, mamíferos) es conocida y los expertos en la técnica la determinan y la llevan a la práctica rutinariamente. La expresión de proteínas por las células cultivadas de acuerdo con los procedimientos de la presente invención, puede situarse bajo el control de promotores tales como promotores víricos, por ejemplo, citomegalovirus (CMV), virus del sarcoma de Rous (RSV), fosfoglicerol quinasa (PGK), timidina quinasa (TK) o el promotor de la α-actina. Además, los promotores regulados confieren inducibilidad mediante compuestos o moléculas particulares, por ejemplo, el elemento de respuesta a glucocorticoides (GRE) del virus del tumor mamario de ratón (MMTV) se induce por glucorticoides (V. Chandler et al., 1983, Cell, 33:489-499). También pueden usarse promotores o elementos reguladores específicos de tejidos (G. Swift et al., 1984, Cell, 38:639-646), si se necesita o
55 se desea.
Pueden introducirse constructos de expresión en las células mediante varios procedimientos de transferencia génica conocidos por los expertos en la técnica, por ejemplo, procedimientos convencionales de transfección génica, tales como coprecipitación con fosfato de calcio, transfección liposómica, microinyección, electroporación e infección o transducción vírica. La elección del procedimiento está dentro de la competencia del experto en la técnica. Para un experto en la técnica, será aparente que uno o más constructos que portan secuencias de ADN para su expresión en células pueden transfectarse a las células de modo que los productos de expresión se producen a continuación en las células y/o se obtienen a partir de las mismas.
65 En un aspecto particular, los sistemas de expresión de mamíferos que contienen secuencias de control y reguladoras apropiadas son preferentes para usar en las células de mamífero que expresan proteínas de la presente
13
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Application Number | Priority Date | Filing Date | Title |
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US897857 | 1997-07-21 | ||
US27834309P | 2009-10-06 | 2009-10-06 | |
US278343P | 2009-10-06 | ||
US12/897,857 US9540426B2 (en) | 2009-10-06 | 2010-10-05 | Mammalian cell culture processes for protein production |
PCT/US2010/051552 WO2011044180A1 (en) | 2009-10-06 | 2010-10-06 | Methods of production of glycoproteins in mammalian cell cultures using glucocorticoids |
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ES2613269T3 true ES2613269T3 (es) | 2017-05-23 |
ES2613269T5 ES2613269T5 (es) | 2020-08-03 |
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ES10765714T Active ES2613269T5 (es) | 2009-10-06 | 2010-10-06 | Métodos para la producción de glucoproteínas en cultivos de células de mamífero usando glucocorticoides |
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US (3) | US9540426B2 (es) |
EP (1) | EP2486048B2 (es) |
JP (1) | JP2013506436A (es) |
KR (1) | KR20120100973A (es) |
CN (1) | CN102753572A (es) |
AR (1) | AR078544A1 (es) |
AU (1) | AU2010303590B2 (es) |
BR (1) | BR112012007854A2 (es) |
CA (1) | CA2777050C (es) |
CO (1) | CO6531436A2 (es) |
CY (1) | CY1118734T1 (es) |
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EA (2) | EA025604B1 (es) |
ES (1) | ES2613269T5 (es) |
HR (1) | HRP20170269T4 (es) |
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LT (1) | LT2486048T (es) |
MX (1) | MX2012003654A (es) |
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RS (1) | RS55727B2 (es) |
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SM (1) | SMT201700116B (es) |
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