WO2012110435A1 - Additifs pour procédé biologique - Google Patents

Additifs pour procédé biologique Download PDF

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Publication number
WO2012110435A1
WO2012110435A1 PCT/EP2012/052366 EP2012052366W WO2012110435A1 WO 2012110435 A1 WO2012110435 A1 WO 2012110435A1 EP 2012052366 W EP2012052366 W EP 2012052366W WO 2012110435 A1 WO2012110435 A1 WO 2012110435A1
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WO
WIPO (PCT)
Prior art keywords
acid
cells
sterically hindered
cell culture
process according
Prior art date
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PCT/EP2012/052366
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English (en)
Inventor
Franz Kaufmann
Andreas Hafner
Said RABBANI
Beat Ernst
Original Assignee
Basf Se
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Publication of WO2012110435A1 publication Critical patent/WO2012110435A1/fr

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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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
    • 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
    • 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
    • C12N2500/30—Organic components
    • 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
    • C12N2511/00—Cells for large scale production

Definitions

  • the present invention relates to biosynthetic processes based on mammalian cell cultures using certain additives for improving the productivity, especially for the preparation of pharmaceutically active proteins such as antibodies and fragments of antibodies, as well as to cell culture media for mammalian cells containing said additive(s).
  • oxidative stress and other radical reactions can lead to damage at molecules such as proteins, nucleic acids or other small or large molecular weight molecules such as lipids, amino acids, sugars or the like, e.g. at double bonds, sulphur etc.
  • Natural antioxidants (Vitamins C & E, polyphenols, amino acids such as cysteine and methionine, peptides such as gluthatione and carnosine) scavenge reactive oxygen species (ROS) and are used in medicine, personal care and nutrition as well as in fermentation processes.
  • ROS reactive oxygen species
  • Nitroxide radicals such as the 2,2,6,6- Tetramethyl-1-piperidinyloxy radical and 4-hydroxy-2,2,6,6-tetramethyl-1-piperidinyloxyl have already been examined in few single cases as additives to cell culture.
  • US 2004/024025 describes that TEMPO causes increased apoptosis in human cancer cells due to SAPK and p38 MAPK signal pathway modulation, leading to protein phosphorylation.
  • results available are rather disencouraging from adding sterically hindered nitroxyls to cell cultures.
  • N-oxyl was also used in cell cultures [US2004/024025] in a concentration of 10mM to prevent apoptosis)
  • N-OH Derivatives at relatively low concentration 5mM
  • US 5,462,946 mentions the use of various nitroxide compounds as antioxidants capable of protecting cells, tissues, organs and whole organisms against the deleterious effects of harmful oxygen-derived species generated during oxidative stress in phar- maceutical formulations. It does not specifically refer to the use in the manufacture of products in cell culture or by biosynthesis in cell free systems. Among many other possible uses, it is mentioned only that they can be used in stabilizing labile chemical compounds which undergo spontaneous degradation by generating free radicals in media which are already present in the media as such.
  • the invention thus primarily pertains to a biosynthetic process or method for the manufacture of a protein in and by a cell culture system comprising mammalian cells in a suitable medium, which process comprises keeping the presence of one or more radical scavenging and/or antioxidative agents selected from the group consisting of steri- cally hindered nitroxyls, sterically hindered hydroxylamines, or salts or adducts thereof, in the culture medium at a level from 1 to 5000, and especially 5 to 5000 micromol (sterically hindered nitrogen) per litre of the cell culture system through a part or all of the biosynthetic manufacturing process.
  • one or more radical scavenging and/or antioxidative agents selected from the group consisting of steri- cally hindered nitroxyls, sterically hindered hydroxylamines, or salts or adducts thereof, in the culture medium at a level from 1 to 5000, and especially 5 to 5000 micro
  • Preferred concentrations of the sterically hindered nitroxyls, sterically hindered hydroxylamines, or salts thereof are from the range 1 to 1500 micromol, especially 5 to 1500 micromol, of the agent (amount of sterically hindered nitrogen) per litre of the cell culture system. Also of importance is the dosage range of 1 to less than 1000 micromol, e.g. 1 to 800 micromol or especially 5 to 600 micromol of the agent (each based on sterically hindered nitrogen) per litre of the cell culture system.
  • the sterically hindered nitroxyls and sterically hindered hydroxylamines are mainly selected from 2,2,6,6-tetraalkylpiperidine compounds, like those of the formula I
  • R is hydrogen, C1-C8alkoxy, oxyl or hydroxy, especially oxyl or hydroxy
  • R' is H, oxo, hydroxy or X-A, where X is O or NH and A is H or stands for a residue of a mono, di, tri or tetrava- lent carboxylic acid or hydroxycarboxylic acid, where further valences of the di, tri or tetravalent carboxylic acid or hydroxycarboxylic acid are free COOH, are anionic car- boxylate counterbalanced by cationic ammonium, alkaline or alkaline earth ions and/or carr further hindered nitroxyl or hydroxylamine moieties of the formula
  • Oxo stands for a double bonded
  • Especially preferred species of the formula I are TEMPO, TEMPOL (see abbreviations explained further below), as well as salts or adducts thereof.
  • a small amount (especially below 5mM) of sterically hindered nitroxides (such as TEMPO) and related compounds such as the corresponding hydroxylamines are effectively enhancing the yield of pharmaceutically relevant proteins.
  • sterically hindered nitroxides such as TEMPO
  • related compounds such as the corresponding hydroxylamines
  • significant effects on DC-SIGN/lgG protein expression of both sterically hindered nitroxides TEMPO and TEMPOL in CHO-K1 cells at concentration below 5mM are observed.
  • Preferred sterically hindered nitroxyls and sterically hindered hydroxylamines are selected from compounds of the formula I wherein R is oxo or hydroxy, X is O and A is H or stands for a residue of acetic acid, glycolic acid, citric acid, lactic acid, oxalic acid, malonic acid, hydroxymalonic acid, succinic acid, maleic acid, glutaric acid, hydroxyglu- taric acid, sebacic acid.
  • Salts, and adducts, of the sterically hindered nitroxyls, sterically hindered hydroxylamines, 2,2,6,6-tetraalkylpiperidine compounds are usually selected from with mineral or organic acids (such as hydrochloric acid, hydrobromic acid, acetic acid, glycolic acid, citric acid, lactic acid, oxalic acid, malonic acid, hydroxymalonic acid, succinic acid, maleic acid, glutaric acid, hydroxyglutaric acid, propane tricarboxylic acid, hydroxypro- pane tricarboxylic acid, ethylenediamine tetraacetic acid, sebacic acid, cinnamic acid, including free carboxylates and/or alkali carboxylates thereof especially in case of di-, tri- and tetracarboxylic acids whose acid functionalities are not fully counterbalanced by
  • the product obtainable according is one or more products selected from the group consisting of antibodies, hormones, vaccines, toxins, enzymes, proteins useful in cancer treatment other than those already mentioned, proteins useful in the treatment of bone disorders other than those already mentioned, batch proteins, interferons, inter- leukins, inerleukin receptor antagonists, blood factors, thrombolytic agents, hematopoietic growth factors, other growth factors, tumor necrosis factor, other proteins, nucleic acids, especially antisense therapeutics, ribozymes, gene therapeutics and siRNAs, and small molecules, especially sugars, amino acids, vitamins, steroids, antibiotics or antibiotics precursors, oligopeptides and lipids, and is obtained in free and/or in salt form; and wherein especially the protein prepared is selected from antibodies, antibody precursors and fragments of antibodies.
  • the process according to the invention is generally carried out using cell culture media known in the art of mammalian cell cultivation.
  • the medium thus often contains one or more further active agents; agents may, for example, be selected from antibiotics, antioxidants and other radical scavengers, such as beta-lactames, glycopeptides, poly- ketides, aminoglycosides, polypeptides, chinolones, sulfonamides, vitamines, benzofu- ranone compounds, nitrones, phenolic antioxidants (see, for example, formula 1 of US- 2008-305055, especially compounds 7-33 of Table 1), natural antioxidants or radical scavengers such as tocopherols, coumestanes (such as compounds described in WO 08/1 10465); especially preferred as further components in the present cell culture media are antibiotics (such as penicillin, streptomycin), tocopherols (such as vitamin E, vitamin A). Further components, such as the above optional constituents, are often present in amounts ranging from 1
  • Mammalian cells for cultivation according to the present process are generally selected from cell lines known in the art for biosynthetic processes; examples are ovary cells, myeloma cells, kidney cells, retina derived cells, B cells, hybridoma cells, more particularly Chinese hamster ovary cells (such as CHO-K1 cells), mouse myeloma cells, baby hamster kidney cells, green monkey kidney cells, human retina derived cells, human embryonic kidney cells, hybridoma cells; especially suitable are Chinese hamster ovary cells, and hybridoma cells (e.g. as known in the art as obtainable by the fusion of antibody-producing cells (B-cells) with myeloma cells).
  • the protein obtained according to the invention may conveniently be converted in a further step into a pharmaceutical or nutraceutical composition, e.g. by admixing it with one or more pharmaceutically and/or nutraceutically acceptable carrier materials.
  • the conversion may be effected immediately from the culture medium, or preferably after workup such as isolation or concentration of the desired protein product by methods known in the art (chromatography, affinity chromatography, centrifugation, lyophiliza- tion etc.).
  • the protein product obtainable according to the invention may be free and/or in form of a salt.
  • the invention includes the use of one or more of the antioxidative agents selected from the group consisting of sterically hindered nitroxyls, sterically hindered hydroxylamines, or salts or adducts thereof, as described above, for enhancing the quantity and/or the quality of the product, especially protein, obtainable in a biosynthetic process of manufacture based on mammalian cells, said use comprising adding said additive(s) into a medium in which a cell culture of mammalian cells is comprised.
  • Most preferred mammalian cells are Chinese hamster ovary cells (such as CHO-K1 cells) and hybridoma cells.
  • the invention further comprises using one or more of said additives within a medium in which a cell culture of said mammalian cells is comprised. Further included is a method of use, or the use, of one or more of the above additives for the reduction of detrimental effects of radicals in the cell fermentation process for the product.
  • the invention further pertains to a mammalian cell culture, typically of cell lines known in the art for biosynthetic processes such as ovary cells, myeloma cells, kidney cells, retina derived cells, B cells, hybridoma cells, more particularly Chinese hamster ovary cells, mouse myeloma cells, baby hamster kidney cells, green monkey kidney cells, human retina derived cells, human embryonic kidney cells, hybridoma cells, in liquid or solid form, which cell culture comprises one or more sterically hindered nitroxyls, sterically hindered hydroxylamines, compounds of formula I , or salts or adducts thereof, without or with addition of one or more other components (such as antibiotics etc.) as described above.
  • a mammalian cell culture typically of cell lines known in the art for biosynthetic processes such as ovary cells, myeloma cells, kidney cells, retina derived cells, B cells, hybridoma cells, more particularly Chinese hamster ova
  • the invention includes a cell culture medium for said mammalian cells, or a main component (like concentrate, kit, carbon source, energy source, or in case of a complex medium the extract, e.g. yeast extract etc.) of said mammalian cell culture medium, which com- prises one or more radical scavenging and/or antioxidative additives as defined above, especially of formula I like TEMPO and/or TEMPOL.
  • a cell culture medium for said mammalian cells or a main component (like concentrate, kit, carbon source, energy source, or in case of a complex medium the extract, e.g. yeast extract etc.) of said mammalian cell culture medium, which com- prises one or more radical scavenging and/or antioxidative additives as defined above, especially of formula I like TEMPO and/or TEMPOL.
  • TEMPOL adduct is the salt comprising 3 molecular units of TEMPOL and 1 molecular unit of 2-hydroxy-propane-1 ,2,3-tricarboxylic acid
  • Tween20 polysorbate 20 polyoxyethylene-sorbitan monolaurate of 20 ethylene units
  • CHO-K1 cells producing DC-SIGN/lgG are cultivated in F-12 nutrient mixture medium (cat. 21765, Gibco) supplemented with 10% FCS (cat. 10500, Lot. 07Q2396K, Gibco), optionally with further addition of penicillin (100 U)/streptomycin (100 ⁇ g/ml) (cat.
  • TEMPO or TEMPOL suspended in the culture medium (75 mM) and sterile filtrated are added to the cells at 10 ⁇ , 100 ⁇ and 1 mM concentrations.
  • TEMPOL adduct is added in 10 mg/L, 100 mg/L, 250 mg/L and 500 mg/L concentrations (corresponding to 42 ⁇ , 421 ⁇ , 1.05 mM and 2.1 mM with respect to TEMPOL).
  • the cells are inspected daily by microscopy.
  • the medium is collected after 1 week of cell culture, filtrated (0.22 ⁇ ), mixed (1 :1) with loading buffer (50 mM tris-HCI pH 7.6, 150 mM NaCI and 0.05% Tween20) and loaded on a protein A column for affinity purification. Two washing steps are performed, the first with 10 bed volumes of loading buffer and the second with 2 bed volumes of 5 mM NH40Ac (pH 5.0).
  • the protein is eluted with 10 mL of 500 mM NH40Ac (pH 3.4).
  • the collected fractions are immediately neutralized with 2.5 M Tris-HCI solution and concentrated by ultrafiltration (Ami- con, YM 50). Results of microscopic inspections:
  • the protein expression in the presence of 10, 100 and 1000 ⁇ concentrations of TEMPO, TEMPOL with or without antibiotics (penicillin/streptomycin) and in the presence of TEMPOL adduct in 10 mg/L, 100 mg/L, 250 mg/L and 500 mg/L concentrations is determined by HPLC.
  • Example 2 The procedure of example 1 is repeated using Hybridoma cells instead of CHO-K1 cells. The results show improved antibody production.
  • Figure 1 Evaluation of DC-SIGN expression in CHO-K1 cells in the presence of
  • FIG. 2 Evaluation of DC-SIGN expression in CHO-K1 cells in the presence of TEMPO and TEMPOL.
  • K positive control
  • T TEMPO
  • TL TEMPOL
  • 10,100 and 1000 ⁇ (-) absence of penicillin/streptomycin in the culture medium.
  • Figure 3 Evaluation of DC-SIGN expression in CHO-K1 cells in the presence of TEM- POL adduct at 10, 100, 250 and 500 mg/L, absence of penicillin/streptomycin in the culture medium.

Abstract

La présente invention concerne un milieu de culture cellulaire pour cellules de mammifère et un procédé correspondant permettant de préparer une protéine selon un procédé de biosynthèse dans un système de culture cellulaire contenant des cellules de mammifère. Le procédé comprend le maintien de la présence d'un ou de plusieurs désactivateurs de radicaux et/ou d'agents antioxydants, choisis parmi les nitroxyles stériquement encombrés et les hydroxylamines stériquement encombrées (et leurs sels ou adduits), dans le milieu de culture à une concentration de 5 à 5000 micromoles (azote stériquement encombré) par litre de système de culture cellulaire pendant une partie ou la totalité du procédé biosynthétique de préparation.
PCT/EP2012/052366 2011-02-14 2012-02-13 Additifs pour procédé biologique WO2012110435A1 (fr)

Applications Claiming Priority (4)

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US201161442305P 2011-02-14 2011-02-14
US61/442,305 2011-02-14
EP11154290.8 2011-02-14
EP11154290 2011-02-14

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8883442B2 (en) 2011-07-13 2014-11-11 Foodchek Systems, Inc. Culture medium, method for culturing Listeria, and method for detecting Listeria
US9221789B2 (en) 2012-06-22 2015-12-29 Basf Se Multicomponent crystals comprising imatinib mesilate and selected co-crystal formers

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4927762A (en) * 1986-04-01 1990-05-22 Cell Enterprises, Inc. Cell culture medium with antioxidant
WO1991013619A1 (fr) * 1990-03-16 1991-09-19 The United States Of America, Represented By The Secretary, United States Department Of Commerce Nitroxydes utilises comme protecteurs contre la contrainte oxydative
US20040024025A1 (en) 1998-06-26 2004-02-05 Georgetown University Compositions and methods for inducing cell death
WO2007046729A1 (fr) * 2005-10-18 2007-04-26 Limited Liability Company 'mitotechnology' Méthode pour agir sur un organisme par livraison ciblée de substances biologiquement actives sur des mitochondries, composition pharmaceutique pour appliquer ladite méthode, et composé à utiliser dans ce but
WO2008033517A2 (fr) * 2006-09-13 2008-03-20 Abbott Laboratories Améliorations à la culture de cellules
WO2008110465A1 (fr) 2007-03-09 2008-09-18 Basf Se Antioxydants de type phytoestrogène et absorbants d'uv
US20080305055A1 (en) 2004-12-22 2008-12-11 Werner Baschong Anti-Radical Agents
WO2011018472A2 (fr) * 2009-08-14 2011-02-17 Basf Se Procédés en cultures cellulaires, et inventions apparentées, utilisant certains additifs

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4927762A (en) * 1986-04-01 1990-05-22 Cell Enterprises, Inc. Cell culture medium with antioxidant
WO1991013619A1 (fr) * 1990-03-16 1991-09-19 The United States Of America, Represented By The Secretary, United States Department Of Commerce Nitroxydes utilises comme protecteurs contre la contrainte oxydative
US5462946A (en) 1990-03-16 1995-10-31 The United States Of America As Represented By The Department Of Health And Human Services Nitroxides as protectors against oxidative stress
US20040024025A1 (en) 1998-06-26 2004-02-05 Georgetown University Compositions and methods for inducing cell death
US20080305055A1 (en) 2004-12-22 2008-12-11 Werner Baschong Anti-Radical Agents
WO2007046729A1 (fr) * 2005-10-18 2007-04-26 Limited Liability Company 'mitotechnology' Méthode pour agir sur un organisme par livraison ciblée de substances biologiquement actives sur des mitochondries, composition pharmaceutique pour appliquer ladite méthode, et composé à utiliser dans ce but
WO2008033517A2 (fr) * 2006-09-13 2008-03-20 Abbott Laboratories Améliorations à la culture de cellules
WO2008110465A1 (fr) 2007-03-09 2008-09-18 Basf Se Antioxydants de type phytoestrogène et absorbants d'uv
WO2011018472A2 (fr) * 2009-08-14 2011-02-17 Basf Se Procédés en cultures cellulaires, et inventions apparentées, utilisant certains additifs

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Title
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CASTAGNA R ET AL: "Nitroxide radical TEMPO reduces ozone-induced chemokine IL-8 production in lung epithelial cells", TOXICOLOGY IN VITRO, ELSEVIER SCIENCE, GB, vol. 23, no. 3, 1 April 2009 (2009-04-01), pages 365 - 370, XP025988330, ISSN: 0887-2333, [retrieved on 20081230], DOI: DOI:10.1016/J.TIV.2008.12.016 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8883442B2 (en) 2011-07-13 2014-11-11 Foodchek Systems, Inc. Culture medium, method for culturing Listeria, and method for detecting Listeria
US9221789B2 (en) 2012-06-22 2015-12-29 Basf Se Multicomponent crystals comprising imatinib mesilate and selected co-crystal formers

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