ES2533266T3 - Purificación de anticuerpos mediante cromatografía de intercambio catiónico - Google Patents

Purificación de anticuerpos mediante cromatografía de intercambio catiónico Download PDF

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ES2533266T3
ES2533266T3 ES08844379.1T ES08844379T ES2533266T3 ES 2533266 T3 ES2533266 T3 ES 2533266T3 ES 08844379 T ES08844379 T ES 08844379T ES 2533266 T3 ES2533266 T3 ES 2533266T3
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cation exchange
nacl
month
purification
exchange chromatography
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ES2533266T5 (es
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Benedicte Andree Lebreton
Deborah Ann O'connor
Aurelia Safta
Mandakini Sharma
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Genentech Inc
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    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/18—Ion-exchange chromatography
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/08—Vasodilators for multiple indications
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/14—Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
    • C07K16/065—Purification, fragmentation
    • 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
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • 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
    • C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
    • 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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Abstract

Un método para purificar un anticuerpo de una composición que comprende el anticuerpo y al menos un contaminante, comprendiendo dicho método las etapas secuenciales de: (a) cargar la composición en un material de intercambio catiónico en donde la composición está a un primer pH de 4,0 a 6,0; (b) lavar el material de intercambio catiónico con un primer tampón de lavado, en donde el pH del primer tampón de lavado es de 6,8 a 9,0; (c) lavar el material de intercambio catiónico con un segundo tampón de lavado de un pH de 5,0 a 6,0; y (d) eluir el anticuerpo del material de intercambio catiónico con un tampón de elución a una conductividad que es al menos 2 mS/cm mayor que la del segundo tampón de lavado.

Description

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E08844379
18-03-2015
Ejemplo 1: Purificación de un anticuerpo contra CD20
Este ejemplo describe un proceso mejorado de cromatografía de intercambio catiónico para purificar un anticuerpo contra CD20, rituximab. Rituximab se usa para terapia de NHL, CLL, RA, MS, etc. La estructura de la molécula de 5 Rituximab se describe en el documento 5.736.137, Anderson et al., así como en las Fig. 1A-1B de este documento. Rituximab está disponible en el mercado en Genentech, Inc.
Se usa cromatografía de intercambio catiónico para reducir adicionalmente los niveles de CHOP, ADN, proteína A lixiviada, garamicina (GENTAMYCIN®), agregados de Rituximab, y virus potenciales. Rituximab se une a la columna
10 en las condiciones de carga. La columna después se lava, eluye, regenera/desinfecta, y almacena hasta el siguiente uso. Pueden usarse múltiples ciclos para procesar un lote completo de combinación de afinidad. La combinación de intercambio catiónico puede mantenerse a temperatura ambiente hasta 30 ºC durante hasta 3 días o a 5 ºC durante hasta 7 días.
15 La resina de intercambio catiónico (POROS 50 HS®, Applied Biosystems) se compacta en una columna hasta una altura de lecho de 17-33 cm. Antes de cargar la combinación de afinidad, se purga la columna de intercambio catiónico de solución de almacenamiento con tampón de equilibrado. Después del equilibrado, la combinación de afinidad se carga en la columna. El producto se une a la columna en estas condiciones. La columna después se lava con tampón de lavado 1, seguido de tampón de lavado 2. Rituximab se eluye de la columna usando un tampón de
20 elución de alta fuerza iónica.
Se proporciona una comparación de las condiciones para el proceso de la presente invención en comparación con el proceso original (control) en la siguiente tabla.
25 Tabla 1: Comparación de tampones para procesos de cromatografía de intercambio catiónico de Rituximab
Fase
Composición de tampón (proceso original) Composición de tampón (proceso ejemplificado)
Pre-equilibrado
MES 20 mM, NaCl 500 mM, pH 5,50 Ninguna
Equilibrado
MES 20 mM, NaCl 60 mM, pH 5,50 MES 19 mM, NaCl 60 mM, pH 5,50
Carga
Combinación de proteína A condicionada, pH 5,00, densidad de carga ≤ 50 g/l de resina Combinación de proteína A condicionada, pH 5,00, densidad de carga ≤ 50 g/l de resina
Lavado 1
MES 20 mM, NaCl 60 mM, pH 5,50 HEPES 25 mM, pH 7,80
Lavado 2
Ninguno MES 19 mM, NaCl 10 mM, pH 5,50
Elución
MES 20 mM, NaCl 160 mM, MES 19 mM, NaCl 160 mM,
pH 5,50
pH 5,50
Regeneración
MES 20 mM, NaCl 500 mM, pH 5,50 Ninguna
Desinfección
NaOH 0,5 N NaOH 0,5 N
Almacenamiento
NaOH 0,1 N NaOH 0,1 N
Los intervalos deseados de pH, conductividad y molaridad para la carga y los tampones en el proceso de rituximab se proporcionan en la siguiente tabla.
Tabla 2: Intervalos preferidos de pH, conductividad y molaridad para el proceso de Rituximab
Tampón
Composición de tampón pH diana Intervalo preferido de molaridad de tampón Intervalo preferido de pH de tampón Intervalo permitido de conductividad para tampones
Equilibrado
MES 19 mM, NaCl 60 mM 5,5 MES 14 -23mM NaCl 50 -70 mM 5,0 -6,0 5,0 -7,2 mS/cm
Carga
Combinación de Proteína A condicionada 5,0 NA 4,5 -5,5 2,5 -5,5 mS/cm
Lavado 1
HEPES 25 mM 7,8 HEPES 15 -35 mM 7,5 -8,1 0,5 -1,5 mS/cm
Lavado 2
MES 19 mM NaCl 10 mM 5,5 MES 14 -23mM NaCl 5 -15 mM 5,0 -6,0 0,6 -2,2 mS/cm
Elución
MES 19 mM NaCl 160 mM 5,5 MES 14 -23 mM NaCl 140 -180 mM 5,3 -5,7 13,4 -17,2 mS/cm
Desinfección
NaOH 0,5 N NA NA NA NA
Almacenamiento
NaOH 0,1 N NA NA NA NA
* Valores de conductividad medidos con compensación de temperatura basándose en una temperatura de 20 ºC y un valor alfa de 1,77.
El proceso ejemplificado para purificación de Rituximab potenció la robustez de la eliminación de proteínas de la célula hospedadora posibilitando una mayor retirada de proteínas de la célula hospedadora en las fases de lavado, provocando niveles inferiores de proteínas de la célula hospedadora en la combinación de producto (combinación de 35 elución) y facilitando la retirada de las impurezas en la posterior etapa corriente abajo. La Fig. 3 ilustra las ventajas
17
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Claims (1)

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ES08844379.1T 2007-10-30 2008-10-29 Purificación de anticuerpos mediante cromatografía de intercambio catiónico Active ES2533266T5 (es)

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US98382507P 2007-10-30 2007-10-30
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EP2565206B1 (en) 2016-03-30
PL2215117T3 (pl) 2015-06-30
PT2215117E (pt) 2015-04-01
PH12013501128A1 (en) 2015-12-02
NZ584839A (en) 2012-07-27
KR20100086015A (ko) 2010-07-29
BRPI0817182A2 (pt) 2015-03-17
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