WO2004005493A1 - Animal protein free media for cultivation of cells - Google Patents

Animal protein free media for cultivation of cells Download PDF

Info

Publication number
WO2004005493A1
WO2004005493A1 PCT/EP2003/007341 EP0307341W WO2004005493A1 WO 2004005493 A1 WO2004005493 A1 WO 2004005493A1 EP 0307341 W EP0307341 W EP 0307341W WO 2004005493 A1 WO2004005493 A1 WO 2004005493A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
hydrolysate
virus
concentration
medium
Prior art date
Application number
PCT/EP2003/007341
Other languages
French (fr)
Inventor
Manfred Reiter
Wolfgang Mundt
Leopold Grillberger
Barbara Kraus
Original Assignee
Baxter International, Inc.
Baxter Healthcare S.A.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Baxter International, Inc., Baxter Healthcare S.A. filed Critical Baxter International, Inc.
Priority to EP20030762657 priority Critical patent/EP1520008B1/en
Priority to CA 2491992 priority patent/CA2491992A1/en
Priority to JP2004518745A priority patent/JP2005532057A/en
Priority to MXPA05000418A priority patent/MXPA05000418A/en
Priority to AU2003249990A priority patent/AU2003249990B2/en
Priority to SI200332208T priority patent/SI1520008T1/en
Priority to ES03762657T priority patent/ES2394085T3/en
Priority to NZ538094A priority patent/NZ538094A/en
Priority to DK03762657T priority patent/DK1520008T3/en
Publication of WO2004005493A1 publication Critical patent/WO2004005493A1/en
Priority to HK06103671A priority patent/HK1083635A1/en

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0018Culture media for cell or tissue culture
    • C12N5/005Protein-free medium
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/12Viral antigens
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/12Viral antigens
    • A61K39/145Orthomyxoviridae, e.g. influenza virus
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/12Viral antigens
    • A61K39/275Poxviridae, e.g. avipoxvirus
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/12Viral antigens
    • A61K39/275Poxviridae, e.g. avipoxvirus
    • A61K39/285Vaccinia virus or variola virus
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12Antivirals
    • A61P31/14Antivirals for RNA viruses
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12Antivirals
    • A61P31/14Antivirals for RNA viruses
    • A61P31/16Antivirals for RNA viruses for influenza or rhinoviruses
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00Drugs for immunological or allergic disorders
    • A61P37/02Immunomodulators
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0018Culture media for cell or tissue culture
    • C12N5/0043Medium free of human- or animal-derived components
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00Specific components of cell culture medium
    • C12N2500/70Undefined extracts
    • C12N2500/74Undefined extracts from fungi, e.g. yeasts
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00Specific components of cell culture medium
    • C12N2500/70Undefined extracts
    • C12N2500/76Undefined extracts from plants
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011Details
    • C12N2710/24011Poxviridae
    • C12N2710/24111Orthopoxvirus, e.g. vaccinia virus, variola
    • C12N2710/24134Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011Details
    • C12N2760/16011Orthomyxoviridae
    • C12N2760/16111Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011Details
    • C12N2770/36011Togaviridae
    • C12N2770/36111Alphavirus, e.g. Sindbis virus, VEE, EEE, WEE, Semliki
    • C12N2770/36134Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02ATECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to animal protein free cell culture media comprising a combination of a soy hydrolysate and a yeast hydrolysate.
  • the invention also relates to animal protein free culture processes, wherein cells can be cultivated, propagated and passaged without animal proteins. These processes are useful in cultivating cells, such as recombinant cells or cells infected with a virus, and for producing biological products by cell culture processes.
  • serum or serum derived substances such as albumin, transferrin or insulin
  • serum or serum derived substances may contain unwanted agents that can contaminate the cultures and the biological products obtained therefrom.
  • human serum derived additives have to be tested for all known viruses, including hepatitis and HIV, that can be transmitted by serum.
  • bovine serum and products derived therefrom, for example trypsin bear the risk of BSE-contamination.
  • all serum-derived products can be contaminated by unknown agents.
  • Simple serum free medium typically includes basal medium, vitamins, amino acids, organic and inorganic salts, and perhaps additional components to make the medium nutritionally complex.
  • Such media often are nutritionally insufficient and must be supplemented with animal-derived protein supplements or recombinant versions of proteins used in cell culture, such as insulin, insulin-like growth factor or other growth factors.
  • Cinatl et al., Cell Biology International 17:885-895 (1993) disclose the development of a media (PFK-1) specific for continuous propagation of VERO cells on a polyvinyl formal (PVF) culture surface.
  • PFK-1 media specific for continuous propagation of VERO cells on a polyvinyl formal (PVF) culture surface.
  • WO 96/15231 discloses serum-free medium composed of a synthetic minimal essential medium and yeast extract for propagation of vertebrate cells and virus production process.
  • a medium formulation composed of a basal cell culture medium comprising a rice peptide and an extract of yeast or an enzymatic digest thereof, and/or a plant lipid for growth of animal cells is disclosed in WO 98/15614.
  • a medium comprising purified soy hydrolysate for the cultivation of recombinant cells is disclosed in WO 01/23527.
  • WO 00/03000 discloses a medium that comprises a soy hydrolysate and a yeast extract, but also requires the presence of recombinant forms of animal proteins, such as growth factors.
  • an animal protein free cell culture medium comprising soy hydrolysate and yeast hydrolysate.
  • the soy hydrolysate can be present in a concentration of at least 0.05% (w/v) and yeast hydrolysate is present in a concentration of at least 0.05% (w/v).
  • the soy hydrolysate can be present in a concentration of less than 1.0% (w/v) and yeast hydrolysate can be present in a concentration of less than 0.3% (w/v).
  • the soy hydrolysate can be present in a concentration of between about 0.2% (w/v) to about 0.6% (w/v) and yeast hydrolysate can be present in a concentration of between about 0.05% (w/v) to about 0.2 % (w/v).
  • yeast hydrolysate can be present in a concentration of between about 0.25% (w/v) to about 0.35% (w/v) and the yeast hydrolysate can be present in a concentration of between about 0.05% (w/v) to about 0.15% (w/v).
  • the soy hydrolysate can be present in a concentration of about 0.3% (w/v) and the yeast hydrolysate can be present in a concentration of about 0.1% (w/v).
  • the medium comprises 3 parts by weight soy hydrolysate to 1 part by weight yeast hydrolysate.
  • the yeast hydrolysate can be a ultrafiltrated purified yeast hydrolysate, wherein at least 40% of said yeast hydrolysate has a molecular weight of less than or equal to 500 Daltons.
  • the soy hydrolysate can be a ultrafiltrated purified soy hydrolysate, wherein at least 40% of said soy hydrolysate has a molecular weight of less than or equal to 500 Daltons.
  • the invention also provides methods of cultivating cultures of cells comprising providing a medium comprising soy hydrolysate at a concentration of about 0.05% (w/v) to about 1% (w/v) and yeast hydrolysate at a concentration of about 0.05% (w/v) to about 0.3% (w/v); and propagating the cells in the medium to form the cell culture.
  • Other concentrations of hydrolysates as exemplified above, also can be employed according to the invention.
  • the cells can be animal cells, such as insect cells, avian cells, mammalian cells, stem cells, and preferably those cells that are used for in vitro virus production.
  • the cells also can be recombinant cells.
  • Exemplary cells include those selected from the group of cells consisting of BSC-1 cells, LLC-MK cells, CV-1 cells, COS-cells, VERO cells, MDBK cells, MDCK cells, CRFK cells, RAF cells, RK-cells, TCMK-1 cells, LLC-PK cells, PK15 cells, LLC- RK cells, MDOK cells, BHK-21 cells, CHO cells, NS-1 cells MRC-5 cells, WI-38 cells, BHK cells, 293 cells and RK-cells.
  • the invention also provides an animal protein free confluent cell culture process, comprising: providing an animal protein free medium comprising soy hydrolysate and yeast hydrolysate; growing the cells in the medium, and passaging and sub-cultivating the cells grown in the medium while in contact with a non-animal- derived protease in order to obtain a confluent cell culture.
  • the cells can be animal cells, recombinant cells and/or cells infected with a virus.
  • the media characteristics and cell types set forth herein are applicable here as well.
  • the invention provides a culture of cells cultivated in an animal protein free medium, wherein the medium comprises soy hydrolysate at a concentration of about 0.05% (w/v) to about 1 % (w/v) and yeast hydrolysate at a concentration of about 0.05% (w/v) to about 0.3% (w/v).
  • concentrations of hydrolysates as exemplified above, also can be employed according to the invention.
  • the cells can be animal cells, recombinant cells and/or cells infected with a virus. The media characteristics and cell types set forth herein are applicable here as well.
  • the invention provides methods for producing viruses, comprising: providing a culture of cells that have been grown in an animal protein free medium comprising soy hydrolysate and a yeast hydrolysate; infecting the cells with a virus; and incubating the infected cells to propagate the virus.
  • the cells can be animal cells and/or recombinant cells and, in particular, mammalian cells.
  • the media characteristics and cell types set forth herein are applicable here as well.
  • the viruses to be produced on the cultured cells may be chosen from the range of viruses known to infect the cultured cell type.
  • viruses when utilizing a mammalian cell culture, viruses may be chosen from the genera of orthomyxoviruses, paramyxoviruses, reoviruses, picornaviruses, flaviviruses, arenaviruses, herpesviruses, poxviruses, coronaviruses and adenoviruses.
  • the virus used may be a wild-type virus, an attenuated virus, a reassortant virus, or a recombinant virus.
  • an infectious nucleic acid clone may be utilized according to infectious clone transfection methods known to those of skill in the field of virology.
  • the invention further provides methods for producing poxviruses (including vaccinia virus), comprising: providing a culture of cells that have been grown in an animal protein free medium comprising soy hydrolysate and yeast hydrolysate; infecting the cells with poxvirus; and incubating the infected cells to propagate poxvirus.
  • the cells can be mammalian cells and/or recombinant cells. The media characteristics and cell types set forth herein are applicable here as well.
  • the invention further provides methods for producing coronaviruses (including the Severe Acute Respiratory Syndrome associated coronavirus), comprising: providing a culture of cells that have been grown in an animal protein free medium comprising soy hydrolysate and yeast hydrolysate; infecting the cells with coronavirus; and incubating the infected cells to propagate coronavirus.
  • the cells can be mammalian cells and/or recombinant cells. The media characteristics and cell types set forth herein are applicable here as well.
  • the invention provides methods for producing orthomyxoviruses, comprising: providing a culture of cells that have been grown in an animal protein free medium comprising soy hydrolysate and yeast hydrolysate; infecting the cells with orthomyxovirus; and incubating the infected cells to propagate orthomyxovirus.
  • the orthomyxovirus can be Influenza A, B or C virus.
  • the cells can be mammalian cells and/or recombinant cells. The media characteristics and cell types set forth herein are applicable here as well.
  • the invention additionally provides methods for producing Ross River virus, comprising providing a culture of cells that have been grown in an animal protein free medium comprising soy hydrolysate and yeast hydrolysate ; infecting the cells with Ross River virus; and incubating the infected cells to propagate Ross River virus.
  • the cells can be mammalian cells and/or recombinant cells. The media characteristics and cell types set forth herein are applicable here as well.
  • the invention also provides methods for producing Flavivirus, comprising providing a culture of cells that have been grown in an animal protein free medium comprising soy hydrolysate and yeast hydrolysate; infecting the cells with Flavivirus; and incubating the infected cells to propagate Flavivirus.
  • the Flavivirus can be selected from the group consisting of Yellow fever virus, and chimeric viruses derived therefrom, Japanese encephalitis virus, Tick-borne encephalitis virus, West nile Virus and Hepatitis C virus.
  • the cells can be animal cells and/or recombinant cells. The media characteristics and cell types set forth herein are applicable here as well.
  • the invention further provides methods of producing immunogenic compositions comprising a viruses or a virus antigens, wherein the method comprises providing a culture of cells that have been grown in an animal protein free medium comprising soy hydrolysate and yeast hydrolysate; infecting the cells with the virus; incubating the infected cells to propagate the virus; harvesting the virus or virus antigen produced; preparing an immunogenic composition from the harvested virus or virus antigen.
  • the cells can be animal cells and/or recombinant cells. The media characteristics and cell types set forth herein are applicable here as well.
  • the harvested virus or virus antigen can be subjected to purification.
  • the invention also provides methods of producing immunogenic compositions comprising a virus or a virus antigen, wherein the method comprises: providing a culture of mammalian cells, wherein the cells are selected from the group of monkey kidney cells, bovine kidney cells, dog kidney cells, pig kidney cells, mouse kidney cells, rat kidney cells, sheep kidney cells, hamster kidney cells and human cells that have been grown in an animal protein free culture medium comprising a soy hydrolysate and a yeast hydrolysate; infecting the cells with a virus selected from the group of orthomyxoviruses, paramyxoviruses, reoviruses, picornaviruses, flaviviruses, arenaviruses, herpesviruses, poxviruses, coronaviruses and adenoviruses; incubating the culture of cells to propagate the virus; harvesting the virus or virus antigen so produced; and preparing an immunogenic composition from the harvested virus or virus antigen.
  • the method comprises: providing a culture of ma
  • the invention provides cultures of cells infected with orthomyxovirus, poxvirus, paramyxovirus, reovirus, picornavirus, flavivirus, arenavirus, herpesvirus, poxvirus or adenovirus, wherein the cells are cultivated in an animal protein free medium, wherein the medium comprises soy hydrolysate and yeast hydrolysate.
  • the soy hydrolysate can be a concentration of about 0.05% (w/v) to about 1 % (w/v) and the yeast hydrolysate can be at a concentration of about 0.05% (w/v) to about 0.3% (w/v).
  • Other concentrations of hydrolysates as exemplified above, also can be employed according to the invention.
  • the invention further provides preparations of orthomyxovirus, paramyxovirus, reovirus, picornavirus, flavivirus, arenavirus, herpesvirus, poxvirus, coronavirus or adenovirus that are free of animal proteins, including recombinantly- produced versions thereof, from the media, wherein the preparation is obtainable by cultivating cells infected with influenza virus in an animal protein free medium, wherein the medium comprises soy hydrolysate and yeast hydrolysate.
  • the soy hydrolysate can be a concentration of about 0.05% (w/v) to about 1 % (w/v) and the yeast hydrolysate can be at a concentration of about 0.05% (w/v) to about 0.3% (w/v).
  • Other concentrations of hydrolysates as exemplified above, also can be employed according to the invention.
  • These viral preparations are suitable for use to make viral antigen and vaccines after further processing.
  • animal protein free medium in its various grammatical forms, refers to a medium that is not supplemented with proteins and protein components from higher multicellular non-plant eukaryotes (that is, vertebrates), that possess the secondary, tertiary and quaternary structures characteristic of the proteins as they occur in nature.
  • Typical proteins that are avoided are those found in serum and serum derived substances, such as albumin, transferrin, insulin and other growth factors.
  • Recombinantly-produced versions of animal proteins, which can contain immunogenic bacterial components also are avoided according to the invention, and are not present in the animal protein free medium of the invention.
  • Animal proteins and protein components are to be distinguished from non-animal proteins, small polypeptides and oligopeptides obtainable from plants (usually about 10-30 amino acids in length), such as the soy bean, and lower eukaryotes, such as yeast.
  • plants usually about 10-30 amino acids in length
  • the media will contain animal proteins shedded or secreted by those cells, including any recombinant proteins expressed by genetically modified cells if such cells are cultivated.
  • animal protein free medium and biological materials and preparations produced therewith, is not to be construed to require the absence of proteins shedded or secreted by cells propagated in the media, but rather refers to a lack of direct supplementation of media with animal proteins and protein components obtained from animal sources or the like produced recombinantly.
  • basal medium in its various grammatical forms, is a synthetic medium, such as DMEM, HAM's F12, Medium 199 or RPMI, or combinations thereof, and others that are known from the literature or are commercially available.
  • every synthetic medium that does not contain animal proteins, can be used in combination with the soy hydrolysate and yeast hydrolysate combination.
  • the basal medium can comprise a number of ingredients, including amino acids, vitamins, organic and inorganic salts, sources of carbohydrate, each ingredient being present in an amount which supports the cultivation of a cell in vitro.
  • DMEM/HAM's F12 (1 :1 ) medium as basal medium can be used.
  • the medium may contain auxiliary substances, such as buffer substances like sodium bicarbonate, oxidation stabilizers, stabilizers to counteract mechanical stress, or protease inhibitors.
  • auxiliary substances such as buffer substances like sodium bicarbonate, oxidation stabilizers, stabilizers to counteract mechanical stress, or protease inhibitors.
  • a nonionic surfactant such as polypropylene glycol (PLURONIC F-61 , PLURONIC F-68, SYNPERONIC F-68, PLURONIC F-71 or PLURONIC F-108) can be added to the medium as a defoaming agent.
  • PLURONIC F-61 polypropylene glycol
  • PLURONIC F-68 polypropylene glycol
  • SYNPERONIC F-68 PLURONIC F-71 or PLURONIC F-108
  • These agents are generally used to protect cells from the negative effects of aeration since, without an addition of a surfactant, the ascending and bursting air bubbles
  • the quantity of nonionic surfactant is preferably between about 0.05 and about 10 g/L, typically between about 0.1 and about 5 g/L.
  • the medium also can contain cyclodextrin or derivatives thereof, typically between about 0.001 g/L and about 1 g/L.
  • the medium comprises soy hydrolysate and yeast hydrolysate, which can be added to a basal medium.
  • hydrolysate includes an enzymatic digest of soy peptone or yeast extract.
  • the hydrolysate can be obtained from a plurality of soy peptone or yeast extract preparations, respectively, which can be further enzymatically digested (for example, by papain), and/or formed by autolysis, thermolysis and/or plasmolysis.
  • Hydrolysates also may be obtained commercially, such as Hy-Soy, Hy- Yeast 412 and Hi-Yeast 444, from sources such as Quest International, Norwich, New York, OrganoTechnie, S.A. France; or Deutsche Hefewerke GmbH, Germany.
  • Sources of yeast extracts also are disclosed in WO 98/15614.
  • Sources of yeast extracts and soy hydrolysates also are disclosed in WO 00/03000.
  • the hydrolysates used in media of the invention are preferably purified from a crude fraction, because impurities which could interfere with efficient cultivation are preferably eliminated during this purification, thereby improving the consistency of the hydrolysate.
  • Purification can be by ultrafiltration or Sephadex chromatography, for example, with Sephadex G25 or Sephadex G10 or equivalent materials, ion-exchange chromatography, affinity chromatography, size exclusion chromatography or "reversed-phase" chromatography. These processes are known in the field. Using these methods, fractions can be selected which contain soy or yeast hydrolysate of defined molecular weight, preferably ⁇ 1000 Daltons, more preferably ⁇ 500 Daltons, still more preferably ⁇ 350 Daltons.
  • At least 90% of the hydrolysate is preferably of a molecular weight of ⁇ 1000 Dalton.
  • the average molecular weights of the soy and yeast hydrolysates are preferably between about 220 and 375 daltons.
  • the pH value of the soy hydrolysate and the yeast hydrolysate should be in the range between about 6.5 and 7.5.
  • the total nitrogen content should be about between 8 and 11 %, preferably, between 9.0 and 10.0% and the ash content ⁇ 18%.
  • An advantageous hydrolysate is characterized by the feature that it has a free amino acids content of between about 5 and 30%. Endotoxin content, if any, should be ⁇ 500 U/g.
  • One medium according to the invention has the following constituency: synthetic minimal medium (DMEM/HAM's F12 (1 :1 ) medium (1-25 g/L), soy hydrolysate (0.5-10 g/L) and yeast hydrolysate (0.5-3g/L), L-glutamine (0.05-1 g/L), NaHC0 3 (0.1-10 g/L).
  • the pH of the medium is between pH 6.8 and 7.6, preferably between pH 7.0 and 7.3.
  • an animal protein free medium of the invention comprising yeast hydrolysate and soy hydrolysate as described herein, cells showed higher growth rate, higher final cell density of the biomass and increased metabolic activity (expressed in oxygen consumption in % per min) compared to medium either comprising soy hydrolysate or yeast hydrolysate alone, even if the final concentration of yeast hydrolysate or soy hydrolysate solely added to the medium is equivalent to the sum of the combined hydrolysate concentration.
  • a final concentration of about 0.4% (w/v) yeast hydrolysate in the medium alone had an inhibitory effect on cell growth and cell density.
  • a medium comprising 0.4% (w/v) or higher concentrations of soy hydrolysate reached no higher cell density than a medium comprising 0.3% (w/v).
  • a medium comprising a combination of soy hydrolysate and yeast hydrolysate in a final total hydrolysate concentration of 0.4% (w/v) showed a significant increase in cell metabolic activity, cell growth and final cell density.
  • the sum of the amount of the soy and yeast hydrolysate in the medium should be between about 0.2 % (w/v) and about 0.6% (w/v) with a higher ratio of soy hydrolysate in the medium compared to yeast hydrolysate.
  • An optimal ratio between soy and yeast hydrolysate should be about 3 : 1 (soy / yeast), respectively.
  • cells means a generic term and encompass the cultivation of individual cells, tissues, organs, insect cells, avian cells, mammalian cells, primary cells, continuous cell lines, stem cells and/or genetically engineered cells, such as recombinant cells expressing a hetereologous polypeptide or protein.
  • Recombinant cells include, for example, CHO cells or BHK cells expressing heterologous polypeptides or proteins, such as a growth factor or a blood factor.
  • Cells often used for the propagation of virus include VERO cells and CV-1 cells.
  • Mammalian cells suitable for cultivation in the cell culture medium of the present invention include those of human origin, which may be primary cells derived from a tissue sample, diploid cell strains, transformed cells or established cell lines. Mammalian cells can include human and non-human cells alike. Mammalian cells of non-human origin can be monkey kidney cells, bovine kidney cells, dog kidney cells, pig kidney cells, rabbit kidney cells, mouse kidney cells, rat kidney cells, sheep kidney cells, hamster kidney cells, Chinese hamster ovarian cells or an animal cell derived from any tissue.
  • mammalian cells that can cultivated in the culture medium can be BSC-1 cells, LLC-MK cells, CV-1 cells, COS- cells, COS-1 cells, COS-3 cells, COS-7 cells, VERO cells, MDBK cells, MDCK cells, CRFK cells, RAF cells, RK-cells, TCMK-1 cells, LLC-PK cells, PK15 cells, LLC-RK cells, MDOK cells, BHK-21 cells, CHO cells, 293 cells, NS-1 cells MRC-5 cells, Wl- 38 cells, BHK cells, 293 cells and RK-cells.
  • Examples or recombinant cells include CHO cells expressing Factor VIII, Fll, FIX, FX, vWF, for example, all of which are known to the person skilled in the art.
  • continuous cells or “continuous cell line” (CCL), in their various grammatical forms, mean cultured cells that replicate indefinitely and are capable of growing in suspension culture or large scale cultivation in bioreactor.
  • the unrestricted growth of CCLs permits long-term cultivation from a standardized cell substrate and low costs.
  • Mammalian cell lines can be selected from the group of CHO cells, COS cells, VERO cells, LLK-MK2 cells, NS-1 cells, MDBK cells, MDCK cells, MRC-5 cells, WI-38 cells, BHK cells, CV-1 cells, rabbit kidney (RK) cells and other cell lines as disclosed by Butler et al. BIOS Scientific Publisher p.1-24 (1992), which is incorporated herein by reference.
  • the CCLs are preferably tested for absence of adventitious agents, such as bacteria, fungi, mycoplasma, protozoans and viruses.
  • cell culture in its various grammatical forms, refers to cells grown in suspension, roller bottles, flasks and the like. Large scale approaches, such as bioreactors, including adherent cells growing attached to microcarriers in stirred fermentors, also are included. Moreover, it is possible to not only to culture surface-dependent cells, but also to use the suspension culture techniques with the inventive media. If the cells are grown on microcarriers, the microcarrier can be selected from the group of microcarriers based on dextran, collagen, plastic, gelatin and cellulose and others as described in Butler, Spier & Griffiths, Animal cell Biotechnology 3:283-303 (1988). Porous carriers, such as e.g.
  • Cytoline® or Cytopore® as well as dextran-based carriers, such as DEAE-dextran (Cytodex 1®), quaternary amine-coated dextran (Cytodex 2®) or gelatin-based carriers, such as gelatin-coated dextran (Cytodex 3®) are suitable. These carriers can be obtained from Pharmacia.
  • the cells are preferably grown from the ampoule to the biomass in the animal protein free media and kept under culture medium conditions during cell culture growth and product production process.
  • Cells that have already been adapted to the media are preferably used. It has been found that not only increased yields be achieved with such pre-adapted cells, but their stability for cultivation also is clearly improved by the use of the medium in accordance with the invention.
  • Mammalian cells are typically cultivated in a cell incubator at about 37°C, with the culture medium having an optimal pH in the range of about 6.8 to 7.6, preferably between 7.0 and 7.3.
  • Cells in batch culture might have a complete medium change about every 2 to 3 days, or more or less frequently, if required.
  • Cells in perfusion culture e.g. in bioreactor or fermenter
  • Cultivation approaches can include, depending on context and need, the sub-cultivation, passaging and propagation of the cells.
  • the invention thus provides methods for cultivating cells comprising the steps of growing cells in a basal medium comprising yeast hydrolysate and soy hydrolysate.
  • the cells are grown in a medium comprising soy hydrolysate in a concentration of 0.05% (w/v) to 1.0 % (w/v) and yeast hydrolysate in a concentration of 0.05% (w/v) to 0.3 % (w/v).
  • the cells are grown from small scale to large scale biomass in animal protein free media of the invention.
  • the passaging and subcultivation of the cells to obtain a cell culture biomass is preferably performed with a non-animal-derived protease, such as Pronase or a purified fraction thereof.
  • the carrier is preferably a synthetic carrier, or a microcarrier coated with a non-animal derived material.
  • a DEAE-dextran or quaternary amine-coated dextran microcarrier for example a DEAE-dextran or quaternary amine-coated dextran microcarrier.
  • the invention also provides an animal protein free cell culture process, wherein cells are cultivated, sub-cultivated and passaged under conditions devoid of animal proteins.
  • the process comprising the steps of providing an animal protein free medium comprising yeast hydrolysate and soy hydrolysate, growing cells in said medium, passaging and sub-cultivating said cells grown in that medium using a non- animal-derived protease, further growing the sub-cultivated cells to reach a confluent cell density and repeating the steps of sub-cultivation and growth of cells until the final cell culture biomass desired is reached.
  • the process includes the growth of the cells in animal protein free medium, sub-cultivating and passaging the cells using a non-animal derived protease, preferably a purified trypsin-like fraction of Streptomyces griseus (SGT).
  • a non-animal derived protease preferably a purified trypsin-like fraction of Streptomyces griseus (SGT).
  • SGT purified trypsin-like fraction of Streptomyces griseus
  • Cells suitable for growth in the animal protein free media of the present invention include, but are not limited to, BSC-1 cells, LLC-MK cells, CV-1 cells, VERO cells, MDBK cells, MDCK cells, CRFK cells, RAF cells, TCMK-1 cells, LLC- PK cells, PK15 cells, LLC-RK cells, MDOK cells, RK-cells, BHK-21 cells, WI-38 cells, 293 cells and/or MRC-5 cells.
  • viruses such as an orthomyxovirus, paramyxovirus, reovirus, picornavirus, flavivirus, arenavirus, herpesvirus, poxvirus, coronavirus, adenovirus and other viruses known to the skilled person.
  • the virus used to infect the cell culture can be Influenza virus, vaccinia virus and variola, fowlpox virus, cowpox virus, tick-borne encephalitis virus (TBE), poliovirus, Hepatitis A Virus, Ross River Virus, Yellow fever virus and a chimeric virus derived thereof, West nile virus, Japanese encephalitis virus, rubella virus, hepatitis C virus (HCV), mumps virus, measles virus, respiratory syncytial virus (RSV), herpes simplex virus (HSV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), rotavirus, foot and mouth disease virus (FMDV).
  • Influenza virus vaccinia virus and variola
  • fowlpox virus cowpox virus
  • tick-borne encephalitis virus TBE
  • poliovirus Hepatitis A Virus
  • Ross River Virus Ross River Virus
  • Yellow fever virus and a chimeric virus derived
  • the cells can be cultivated in the media of the invention and grown to reach an optimal cell density prior infection with the respective virus.
  • a cell culture grown and propagated in an animal protein free medium of the invention shows a significant increase of virus yield productivity.
  • Examples of different viruses propagated on cells cultivated and grown of the medium of the invention have shown a 2 to 5 fold increase of virus yield compared to a medium comprising solely yeast extract. This makes the system more favorable for cell growth and virus production processes than the those described in the prior art.
  • the cells are VERO cells and the virus is selected from the group of Influenza Virus, TBE-Virus, vaccinia virus, poliovirus, Hepatitis A Virus, Ross River Virus, Yellow fever virus and a chimeric virus derived thereof, West nile virus, Japanese encephalitis virus, rubella virus, HCV, mumps virus, measles virus, respiratory syncytial virus, HSV, CMV, EBV, rotavirus.
  • Other viruses known to grow in VERO cells also can be used.
  • the invention also provides for production of vaccinia virus by providing a culture of cells grown and cultivated in an animal protein free medium comprising yeast hydrolysate and soy hydrolysate, infecting said cells with a vaccinia virus and incubating the culture of cells to propagate the vaccinia virus.
  • the cells are grown in a medium comprising soy hydrolysate in a concentration of about 0.05% (w/v) to about 1.0% (w/v) and yeast hydrolysate concentration of about 0.05% (w/v) to about 0.3% (w/v).
  • the cells can VERO cells, CV-1 cells, RK-cells, BHK-21 cells, MRC-5 cells, or any cell to which vaccinia virus can be grown.
  • the vaccinia virus can be a naturally-occurring vaccinia virus, smallpox virus vaccine strain, virulent vaccinia strains, attenuated vaccinia strains and a recombinant vaccinia viruses.
  • orthomyxovirus by providing a culture of cells cultivated and grown in an animal protein free medium made from a basal medium comprising yeast hydrolysate and soy hydrolysate, infecting the cells with a orthomyxovirus and incubating the culture of cells to propagate the orthomyxovirus.
  • the cells are grown in a medium comprising soy hydrolysate in a concentration of about 0.05% (w/v) to about 1.0 % (w/v) and yeast hydrolysate in a concentration of about 0.05% (w/v) to about 0.3 % (w/v).
  • the cells can be BSC-1 cells, CV-1 cells, VERO cells, MDBK cells, MDCK cells, MDOK cells, BHK-21 cells, WI-38 cells, MRC-5 cells or any cell to which orthomyxovirus can be propagated.
  • the orthomyxovirus can be a Influenza virus, such as Influenza A, B or C.
  • the cells can be BSC- 1 cells, CV-1 cells, VERO cells, MDBK cells, MDCK cells, CRFK cells, BHK-21 cells, WI-38 cells, MRC-5 cells or any cell to which Ross River Virus can be propagated.
  • Flavivirus by providing a culture of cells grown and cultivated on an animal protein free medium made from a basal medium comprising yeast hydrolysate and soy hydrolysate, infecting the cells with a Flavivirus and incubating the culture of cells to propagate the Flavivirus.
  • the cells are grown in a medium comprising soy hydrolysate in a concentration of about 0.05% (w/v) to about 1.0 % (w/v) and yeast hydrolysate in a concentration of about 0.05% (w/v) to about 0.3 % (w/v).
  • the Flavivirus can be a Yellow fever virus, or a recombinant of chimeric derivatives thereof, Japanese encephalitis virus, Tick-borne encephalitis virus, West nile Virus, and Hepatitis C virus.
  • the cells types identified herein can be used for the propagation of flavivirus.
  • Picornavirus by providing a culture of cells grown and cultivated on an animal protein free medium made from a basal medium comprising yeast hydrolysate and soy hydrolysate, infecting the cells with a Picornavirus and incubating the culture of cells to propagate the Picornavirus.
  • the cells are grown in a medium comprising soy hydrolysate in a concentration of about 0.05% (w/v) to about 1.0 % (w/v) and yeast hydrolysate in a concentration of about 0.05% (w/v) to about 0.3 % (w/v).
  • the Picornavirus can be a poliovirus or hepatitis A virus.
  • the cells types identified herein can be used for the propagation of Picornavirus.
  • the invention also provides methods of producing immunogenic compositions comprising a virus or a virus antigen comprising the steps of providing a culture of animal cells, wherein the cells are selected from the group of monkey kidney cells, bovine kidney cells, dog kidney cells, pig kidney cells, mouse kidney cells, rat kidney cells, sheep kidney cells, rabbit kidney cells, hamster kidney cells and human cells that have been grown the a medium of the invention; infecting the cells with a virus selected from the group of orthomyxoviruses, paramyxoviruses, reoviruses, picornaviruses, flaviviruses, arenaviruses, herpesviruses, poxviruses, coronaviruses and adenoviruses, incubating the culture of cells to propagate the virus, harvesting the virus produced and preparing an immunogenic composition from the virus harvested.
  • the virus produced and harvested can be purified with a method known in the art, such as ion exchange or gel filtration.
  • Animal protein free medium is prepared with a basal DMEM/ HAM's F12 (1 :1 ) medium that is supplemented with inorganic salts, amino acids, vitamins and other components. Also added are sodium bicarbonate (1-3 g/ L), L-Glutamine (0.1 to 1 g /L) and varying concentrations of soy hydrolysate (Quest Technologies, New York) or yeast hydrolysate (Deutsche Hefewerke, Germany) or combinations thereof.
  • EXAMPLE 2 EXAMPLE 2
  • VERO cells African Green Monkey, Cercopthecus aethiops, kidney
  • the cells have been obtained from the American Type Cell Culture Collection, Rockville, Maryland at a passage number 124 under the designation ATCC CCL 81. The cells were grown in various media as described herein.
  • the cell number of the biomass of the cell culture at the end of biomass production was determined either by trypsinization of the cells and counting with a CASY® cell counter (method A) as described by Scharfe et al. Biotechnologie in LaborPraxis 10:1096-1103 1988) or by citric acid and crystal violet treatment followed by counting with a haemocytometer (method B) as described by Sanford et al., J. Nat'l Cancer Inst. 11 :773-795 (1951).
  • VERO cells were cultivated and grown in animal protein free medium comprising yeast hydrolysate in a concentration of 0.05%, 0.1 %, 0.2%, 0.3% or 0.4%, 0.5% (w/v), or soy hydrolysate in a concentration of 0.05%, 0.1 %, 0.2%, 0.3% or 0.4%, 0.5% (w/v), or soy hydrolysate and yeast hydrolysate in a concentration of yeast to soy (yeast / soy) of 0.05% / 0.05% (w/v), 0.1 % / 0.2% (w/v), 0.1 % / 0.3% (w/v), 0.2% / 0.2% (w/v), 0.3% / 0.2% (w/v) or 0.2% / 1.0% (w/v).
  • the cell density of the cell culture at the end of biomass production in animal protein free medium comprising varying concentrations of soy hydrolysate, yeast hydrolysate or combinations thereof was calculated by methods A and B.
  • yeast and soy hydrolysate alone supported cell growth.
  • a concentration of 0.1 % yeast hydrolysate a cell density of about 11.8 x 10 5 cells/ ml was reached, however increasing concentration of yeast hydrolysate to higher than 0.3% (w/v) had a negative effect on cell growth and consequently on cell density.
  • Concentrations of soy hydrolysate alone between 0.1 % (w/v) to 0.2% (w/v) showed less cell growth and cell density than with soy concentrations of 0.3% (w/v) and 0.4% (w/v).
  • soy hydrolysate did not differ significantly and higher concentrations to about 1% w/v of soy hydrolysate had no positive effect on cell growth.
  • the optimal concentration of soy hydrolysate alone was determined to be between 0.2% w/v to 1.0% w/v.
  • the cell density reached at a concentration of 0.05% (w/v) soy and 0.05% (w/v) yeast was about 12.1 x 10 5 cells /ml and had a higher cell culture cell density compared to cells grown in medium comprising solely either 0.1 % (w/v) soy hydrolysate (10 x 10 5 cells/ml) or 0.1% (w/v) yeast hydrolysate (11.8 x 10 5 cells/ml).
  • the cell density of a cell culture grown in a medium comprising yeast with a concentration of 0.2% (w/v) and soy of 1.0% (w/v) was similar to the density obtained in medium comprising 0.05% (w/v) soy and 0.05% yeast (w/v).
  • the metabolic activity of cells cultivated in medium comprising yeast and soy hydrolysate also was higher compared to cells grown in medium solely comprising yeast or soy hydrolysate.
  • the oxygen consumption rate was 1.5 (% per min.) in a medium comprising 0.1 % (w/v) yeast hydrolysate and less than 1.0 (% per min.) in medium comprising 0.4% (w/v) yeast hydrolysate or soy hydrolysate alone.
  • the oxygen consumption was about 2.9 (% per min.), which was about 2 times higher than of cells cultivated in a medium comprising solely soy or yeast hydrolysate.
  • a cell culture of recombinant mammalian cells such as rFVIII-CHO cells, are cultivated in a 10L stirred tank with perfusion.
  • a medium in accordance with Example 1 is used as cultivation and growth medium.
  • the cells are immobilized on a porous microcarrier (Cytopore®, Pharmacia) and cultivated for at least 6 weeks.
  • the perfusion rate is 4 volume changes per day; the pH is 6.9 to 7.2; the 0 2 concentration is approximately 20-50% and the temperature is 37°C.
  • the cell density is determined.
  • VERO cells with a defined passage number were thawed from liquid nitrogen and passaged in roux and roller bottles to produce sufficient cells to inoculate a 1.5 liter bioreactor.
  • Cells are either grown in basal medium supplemented either with yeast hydrolysate or with a combination of yeast hydrolysate and soy hydrolysate as described in Examples 1 and 2. After reaching confluency with a final cell density of 1.5 x 10 6 cells / ml, the cells were released from the microcarrier with a purified fraction of Pronase, the S.
  • griseus trypsin SGT as described in U.S. application serial no. 10/006,223 and transferred to a 10 liter bioreactor. This in turn was used as an inoculum for a 100 liter bioreactor having a microcarrier concentration of 3.0 g/l.
  • SGT griseus trypsin
  • the cells were grown at 37°C.
  • the culture conditions of oxygen saturation 20% +/- 10% and pH 7.1 +/- 0.35 were kept constant during virus propagation process.
  • VERO cells were infected with two different influenza strains, New Caledonia A/H1 N1 and Panama A/H3N2, and propagated in the respective medium. At the end of the virus propagation process the clarified supernatant containing the virus was purified by ultracentrifugation.
  • the harvests of the VERO cells culture with either solely yeast hydrolysate or with yeast and soy hydrolysate were compared on the basis of the volumetric antigen productivity (total SRD, single radial immunodiffusion) and the antigen content of the supernatant at the end of the run (density gradient purified antigen). The yields for both media formulations were compared and are summarized in Table 1. [65] Table 1. Comparison of product yield from VERO Influenza production in different media composition
  • yeast hydrolysate and soy hydrolysate shows a marked improvement over yeast hydrolysate alone.
  • VERO cells were infected with a smallpox vaccine production strain (Dryvax, Wyeth Vaccines, obtained from Acambis, Inc., a calf-lymph vaccine strain adapted for growth in permanent cell line) adapted to growth in animal protein free VERO cells by serial passaging at a multiplicity of infection (m.o.i.) of 0.1 - 0.3. After an incubation time of 2-4 days at 37°C the cells were harvested and virus was recovered from the cells.
  • a smallpox vaccine production strain (Dryvax, Wyeth Vaccines, obtained from Acambis, Inc., a calf-lymph vaccine strain adapted for growth in permanent cell line) adapted to growth in animal protein free VERO cells by serial passaging at a multiplicity of infection (m.o.i.) of 0.1 - 0.3. After an incubation time of 2-4 days at 37°C the cells were harvested and virus was recovered from the cells.
  • Table 2 shows the results of virus yield obtained of cells grown in basal medium supplemented with yeast hydrolysate alone or with yeast and soy hydrolysate. [68] TABLE 2: Determination of Vaccinia virus titer at the end of the production cycle in the bioreactor system.
  • yeast hydrolysate and soy hydrolysate shows a marked improvement over yeast hydrolysate alone.
  • VERO cell culture obtained as described herein were infected with Ross River Virus at a multiplicity of infection (m.o.i.) of 0.1 - 0.3. After an incubation time of 2-4 days at 37°C, the cells were harvested and virus was recovered from the cells.
  • Table 3 shows the results of virus yield obtained of cells grown in basal medium supplemented with yeast hydrolysate alone or with yeast and soy hydrolysate.
  • yeast hydrolysate and soy hydrolysate shows a marked improvement over yeast hydrolysate alone.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Virology (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Pulmonology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The present invention relates to animal protein free cell culture media comprising a combination of non-animal derived peptides derived from soy hydrolysate and yeast hydrolysate. The invention also provides an animal protein free culture process, wherein cells are cultivated, propagated and passaged without animal-derived components. This process is useful for cultivating cells, such as recombinant cells or cells infected with a virus, and for production of biological products by cell culture processes under conditions devoid of animal protein components.

Description

ANIMAL PROTEIN FREE MEDIA FOR CULTIVATION OF CELLS
FIELD OF THE INVENTION
[01] The present invention relates to animal protein free cell culture media comprising a combination of a soy hydrolysate and a yeast hydrolysate. The invention also relates to animal protein free culture processes, wherein cells can be cultivated, propagated and passaged without animal proteins. These processes are useful in cultivating cells, such as recombinant cells or cells infected with a virus, and for producing biological products by cell culture processes.
BACKGROUND OF THE INVENTION
[02] For cultivation of cells, particularly eukaryotic cells, and more specifically mammalian cells, there is a constant need to use special culture media that make available the nutrient substances and growth nutrient substances that are required for efficient growth of the cells and for the production of the proteins or viruses that are desired. Cell culture media formulations have been supplemented with a range of additives, including undefined components like fetal calf serum (FCS), several animal-derived proteins and/or protein hydrolysates of bovine origin.
[03] In general, serum or serum derived substances, such as albumin, transferrin or insulin, may contain unwanted agents that can contaminate the cultures and the biological products obtained therefrom. Furthermore, human serum derived additives have to be tested for all known viruses, including hepatitis and HIV, that can be transmitted by serum. Moreover, bovine serum and products derived therefrom, for example trypsin, bear the risk of BSE-contamination. In addition, all serum-derived products can be contaminated by unknown agents. In the case of serum or protein additives that are derived from human or other animal sources in cell culture, numerous problems (e.g., the varying quality and composition of different batches and the risk of contamination with mycoplasma, viruses or BSE agents) exist, particularly if the cells are used for production of medicinal agents or vaccines for human administration.
[04] Therefore, many attempts have been made to provide efficient host systems and cultivation conditions that do not require serum or other animal protein compounds. Simple serum free medium typically includes basal medium, vitamins, amino acids, organic and inorganic salts, and perhaps additional components to make the medium nutritionally complex. Such media, however, often are nutritionally insufficient and must be supplemented with animal-derived protein supplements or recombinant versions of proteins used in cell culture, such as insulin, insulin-like growth factor or other growth factors.
[05] To avoid the use of animal protein supplements in serum free cell culture medium, several attempts have been made to provide cell culture media that are completely free of proteins.
[06] Cinatl et al., Cell Biology International 17:885-895 (1993) disclose the development of a media (PFK-1) specific for continuous propagation of VERO cells on a polyvinyl formal (PVF) culture surface.
[07] WO 96/15231 discloses serum-free medium composed of a synthetic minimal essential medium and yeast extract for propagation of vertebrate cells and virus production process.
[08] A medium formulation composed of a basal cell culture medium comprising a rice peptide and an extract of yeast or an enzymatic digest thereof, and/or a plant lipid for growth of animal cells is disclosed in WO 98/15614.
[09] A medium comprising purified soy hydrolysate for the cultivation of recombinant cells is disclosed in WO 01/23527.
[10] WO 00/03000 discloses a medium that comprises a soy hydrolysate and a yeast extract, but also requires the presence of recombinant forms of animal proteins, such as growth factors.
[11] For efficient production of biological products, such as viruses or recombinant proteins, it is important that optimal cell density is reached to obtain maximal product yield.
[12] Therefore, a current need exists to increase growth, metabolic activity and density of cells, and to provide optimal cell culture medium devoid of animal proteins for production of biological products, such as those used as medicinals or vaccines in humans. Furthermore, the down-stream processing, e.g. purification of the desired product from culture medium can be more cost-effective and time- efficient if animal proteins are not present in the medium. Additionally, unwanted immunogenic animal proteins may induce deleterious immunological reactions, which are avoided with practice of the present invention.
BRIEF SUMMARY OF THE INVENTION
[13] It is an object of the present invention to provide animal protein free culture media. In accomplishing this and other objects, there is provided, in accordance with one aspect of the invention, an animal protein free cell culture medium comprising soy hydrolysate and yeast hydrolysate. The soy hydrolysate can be present in a concentration of at least 0.05% (w/v) and yeast hydrolysate is present in a concentration of at least 0.05% (w/v). Optionally, the soy hydrolysate can be present in a concentration of less than 1.0% (w/v) and yeast hydrolysate can be present in a concentration of less than 0.3% (w/v). Optionally, the soy hydrolysate can be present in a concentration of between about 0.2% (w/v) to about 0.6% (w/v) and yeast hydrolysate can be present in a concentration of between about 0.05% (w/v) to about 0.2 % (w/v). Optionally, the soy hydrolysate can be present in a concentration of between about 0.25% (w/v) to about 0.35% (w/v) and the yeast hydrolysate can be present in a concentration of between about 0.05% (w/v) to about 0.15% (w/v). Optionally, the soy hydrolysate can be present in a concentration of about 0.3% (w/v) and the yeast hydrolysate can be present in a concentration of about 0.1% (w/v). Optionally, the medium comprises 3 parts by weight soy hydrolysate to 1 part by weight yeast hydrolysate. The yeast hydrolysate can be a ultrafiltrated purified yeast hydrolysate, wherein at least 40% of said yeast hydrolysate has a molecular weight of less than or equal to 500 Daltons. Similarly, the soy hydrolysate can be a ultrafiltrated purified soy hydrolysate, wherein at least 40% of said soy hydrolysate has a molecular weight of less than or equal to 500 Daltons. [14] The invention also provides methods of cultivating cultures of cells comprising providing a medium comprising soy hydrolysate at a concentration of about 0.05% (w/v) to about 1% (w/v) and yeast hydrolysate at a concentration of about 0.05% (w/v) to about 0.3% (w/v); and propagating the cells in the medium to form the cell culture. Other concentrations of hydrolysates, as exemplified above, also can be employed according to the invention. The cells can be animal cells, such as insect cells, avian cells, mammalian cells, stem cells, and preferably those cells that are used for in vitro virus production. The cells also can be recombinant cells. Exemplary cells include those selected from the group of cells consisting of BSC-1 cells, LLC-MK cells, CV-1 cells, COS-cells, VERO cells, MDBK cells, MDCK cells, CRFK cells, RAF cells, RK-cells, TCMK-1 cells, LLC-PK cells, PK15 cells, LLC- RK cells, MDOK cells, BHK-21 cells, CHO cells, NS-1 cells MRC-5 cells, WI-38 cells, BHK cells, 293 cells and RK-cells.
[15] The invention also provides an animal protein free confluent cell culture process, comprising: providing an animal protein free medium comprising soy hydrolysate and yeast hydrolysate; growing the cells in the medium, and passaging and sub-cultivating the cells grown in the medium while in contact with a non-animal- derived protease in order to obtain a confluent cell culture. The cells can be animal cells, recombinant cells and/or cells infected with a virus. The media characteristics and cell types set forth herein are applicable here as well.
[16] Moreover, the invention provides a culture of cells cultivated in an animal protein free medium, wherein the medium comprises soy hydrolysate at a concentration of about 0.05% (w/v) to about 1 % (w/v) and yeast hydrolysate at a concentration of about 0.05% (w/v) to about 0.3% (w/v). Other concentrations of hydrolysates, as exemplified above, also can be employed according to the invention. The cells can be animal cells, recombinant cells and/or cells infected with a virus. The media characteristics and cell types set forth herein are applicable here as well.
[17] Additionally, the invention provides methods for producing viruses, comprising: providing a culture of cells that have been grown in an animal protein free medium comprising soy hydrolysate and a yeast hydrolysate; infecting the cells with a virus; and incubating the infected cells to propagate the virus. The cells can be animal cells and/or recombinant cells and, in particular, mammalian cells. The media characteristics and cell types set forth herein are applicable here as well. The viruses to be produced on the cultured cells may be chosen from the range of viruses known to infect the cultured cell type. For instance, when utilizing a mammalian cell culture, viruses may be chosen from the genera of orthomyxoviruses, paramyxoviruses, reoviruses, picornaviruses, flaviviruses, arenaviruses, herpesviruses, poxviruses, coronaviruses and adenoviruses. The virus used may be a wild-type virus, an attenuated virus, a reassortant virus, or a recombinant virus. In addition, instead of actual virions being used to infect the cells with a virus, an infectious nucleic acid clone may be utilized according to infectious clone transfection methods known to those of skill in the field of virology.
[18] The invention further provides methods for producing poxviruses (including vaccinia virus), comprising: providing a culture of cells that have been grown in an animal protein free medium comprising soy hydrolysate and yeast hydrolysate; infecting the cells with poxvirus; and incubating the infected cells to propagate poxvirus. The cells can be mammalian cells and/or recombinant cells. The media characteristics and cell types set forth herein are applicable here as well.
[19] Furthermore, the invention further provides methods for producing coronaviruses (including the Severe Acute Respiratory Syndrome associated coronavirus), comprising: providing a culture of cells that have been grown in an animal protein free medium comprising soy hydrolysate and yeast hydrolysate; infecting the cells with coronavirus; and incubating the infected cells to propagate coronavirus. The cells can be mammalian cells and/or recombinant cells. The media characteristics and cell types set forth herein are applicable here as well.
[20] Furthermore, the invention provides methods for producing orthomyxoviruses, comprising: providing a culture of cells that have been grown in an animal protein free medium comprising soy hydrolysate and yeast hydrolysate; infecting the cells with orthomyxovirus; and incubating the infected cells to propagate orthomyxovirus. The orthomyxovirus can be Influenza A, B or C virus. The cells can be mammalian cells and/or recombinant cells. The media characteristics and cell types set forth herein are applicable here as well. [21] The invention additionally provides methods for producing Ross River virus, comprising providing a culture of cells that have been grown in an animal protein free medium comprising soy hydrolysate and yeast hydrolysate ; infecting the cells with Ross River virus; and incubating the infected cells to propagate Ross River virus. The cells can be mammalian cells and/or recombinant cells. The media characteristics and cell types set forth herein are applicable here as well.
[22] The invention also provides methods for producing Flavivirus, comprising providing a culture of cells that have been grown in an animal protein free medium comprising soy hydrolysate and yeast hydrolysate; infecting the cells with Flavivirus; and incubating the infected cells to propagate Flavivirus. The Flavivirus can be selected from the group consisting of Yellow fever virus, and chimeric viruses derived therefrom, Japanese encephalitis virus, Tick-borne encephalitis virus, West nile Virus and Hepatitis C virus. The cells can be animal cells and/or recombinant cells. The media characteristics and cell types set forth herein are applicable here as well.
[23] The invention further provides methods of producing immunogenic compositions comprising a viruses or a virus antigens, wherein the method comprises providing a culture of cells that have been grown in an animal protein free medium comprising soy hydrolysate and yeast hydrolysate; infecting the cells with the virus; incubating the infected cells to propagate the virus; harvesting the virus or virus antigen produced; preparing an immunogenic composition from the harvested virus or virus antigen. The cells can be animal cells and/or recombinant cells. The media characteristics and cell types set forth herein are applicable here as well. The harvested virus or virus antigen can be subjected to purification.
[24] The invention also provides methods of producing immunogenic compositions comprising a virus or a virus antigen, wherein the method comprises: providing a culture of mammalian cells, wherein the cells are selected from the group of monkey kidney cells, bovine kidney cells, dog kidney cells, pig kidney cells, mouse kidney cells, rat kidney cells, sheep kidney cells, hamster kidney cells and human cells that have been grown in an animal protein free culture medium comprising a soy hydrolysate and a yeast hydrolysate; infecting the cells with a virus selected from the group of orthomyxoviruses, paramyxoviruses, reoviruses, picornaviruses, flaviviruses, arenaviruses, herpesviruses, poxviruses, coronaviruses and adenoviruses; incubating the culture of cells to propagate the virus; harvesting the virus or virus antigen so produced; and preparing an immunogenic composition from the harvested virus or virus antigen.
[25] Additionally, the invention provides cultures of cells infected with orthomyxovirus, poxvirus, paramyxovirus, reovirus, picornavirus, flavivirus, arenavirus, herpesvirus, poxvirus or adenovirus, wherein the cells are cultivated in an animal protein free medium, wherein the medium comprises soy hydrolysate and yeast hydrolysate. The soy hydrolysate can be a concentration of about 0.05% (w/v) to about 1 % (w/v) and the yeast hydrolysate can be at a concentration of about 0.05% (w/v) to about 0.3% (w/v). Other concentrations of hydrolysates, as exemplified above, also can be employed according to the invention.
[26] The invention further provides preparations of orthomyxovirus, paramyxovirus, reovirus, picornavirus, flavivirus, arenavirus, herpesvirus, poxvirus, coronavirus or adenovirus that are free of animal proteins, including recombinantly- produced versions thereof, from the media, wherein the preparation is obtainable by cultivating cells infected with influenza virus in an animal protein free medium, wherein the medium comprises soy hydrolysate and yeast hydrolysate. The soy hydrolysate can be a concentration of about 0.05% (w/v) to about 1 % (w/v) and the yeast hydrolysate can be at a concentration of about 0.05% (w/v) to about 0.3% (w/v). Other concentrations of hydrolysates, as exemplified above, also can be employed according to the invention. These viral preparations are suitable for use to make viral antigen and vaccines after further processing.
[27] These and other aspects of the invention will become apparent to the skilled person in view of the explanation and data set forth below.
DETAILED DESCRIPTION OF THE INVENTION [28] The term "animal protein free medium," in its various grammatical forms, refers to a medium that is not supplemented with proteins and protein components from higher multicellular non-plant eukaryotes (that is, vertebrates), that possess the secondary, tertiary and quaternary structures characteristic of the proteins as they occur in nature. Typical proteins that are avoided are those found in serum and serum derived substances, such as albumin, transferrin, insulin and other growth factors. Recombinantly-produced versions of animal proteins, which can contain immunogenic bacterial components, also are avoided according to the invention, and are not present in the animal protein free medium of the invention. Animal proteins and protein components are to be distinguished from non-animal proteins, small polypeptides and oligopeptides obtainable from plants (usually about 10-30 amino acids in length), such as the soy bean, and lower eukaryotes, such as yeast. Of course, once the media is contacted or inoculated with the cells to be propagated, the media will contain animal proteins shedded or secreted by those cells, including any recombinant proteins expressed by genetically modified cells if such cells are cultivated. Thus, the term animal protein free medium, and biological materials and preparations produced therewith, is not to be construed to require the absence of proteins shedded or secreted by cells propagated in the media, but rather refers to a lack of direct supplementation of media with animal proteins and protein components obtained from animal sources or the like produced recombinantly.
[29] The term "basal medium," in its various grammatical forms, is a synthetic medium, such as DMEM, HAM's F12, Medium 199 or RPMI, or combinations thereof, and others that are known from the literature or are commercially available. In accordance with the invention, every synthetic medium, that does not contain animal proteins, can be used in combination with the soy hydrolysate and yeast hydrolysate combination. The basal medium can comprise a number of ingredients, including amino acids, vitamins, organic and inorganic salts, sources of carbohydrate, each ingredient being present in an amount which supports the cultivation of a cell in vitro. For example, DMEM/HAM's F12 (1 :1 ) medium as basal medium can be used. The medium may contain auxiliary substances, such as buffer substances like sodium bicarbonate, oxidation stabilizers, stabilizers to counteract mechanical stress, or protease inhibitors. If required, a nonionic surfactant, such as polypropylene glycol (PLURONIC F-61 , PLURONIC F-68, SYNPERONIC F-68, PLURONIC F-71 or PLURONIC F-108) can be added to the medium as a defoaming agent. These agents are generally used to protect cells from the negative effects of aeration since, without an addition of a surfactant, the ascending and bursting air bubbles can lead to damage of those cells that are located on the surface of these air bubbles ("sparging"). The quantity of nonionic surfactant is preferably between about 0.05 and about 10 g/L, typically between about 0.1 and about 5 g/L. In addition, the medium also can contain cyclodextrin or derivatives thereof, typically between about 0.001 g/L and about 1 g/L.
[30] According to the invention, the medium comprises soy hydrolysate and yeast hydrolysate, which can be added to a basal medium. The term "hydrolysate" includes an enzymatic digest of soy peptone or yeast extract. The hydrolysate can be obtained from a plurality of soy peptone or yeast extract preparations, respectively, which can be further enzymatically digested (for example, by papain), and/or formed by autolysis, thermolysis and/or plasmolysis. Hydrolysates also may be obtained commercially, such as Hy-Soy, Hy- Yeast 412 and Hi-Yeast 444, from sources such as Quest International, Norwich, New York, OrganoTechnie, S.A. France; or Deutsche Hefewerke GmbH, Germany. Sources of yeast extracts also are disclosed in WO 98/15614. Sources of yeast extracts and soy hydrolysates also are disclosed in WO 00/03000.
[31] The hydrolysates used in media of the invention are preferably purified from a crude fraction, because impurities which could interfere with efficient cultivation are preferably eliminated during this purification, thereby improving the consistency of the hydrolysate. Purification can be by ultrafiltration or Sephadex chromatography, for example, with Sephadex G25 or Sephadex G10 or equivalent materials, ion-exchange chromatography, affinity chromatography, size exclusion chromatography or "reversed-phase" chromatography. These processes are known in the field. Using these methods, fractions can be selected which contain soy or yeast hydrolysate of defined molecular weight, preferably <1000 Daltons, more preferably <500 Daltons, still more preferably <350 Daltons. At least 90% of the hydrolysate is preferably of a molecular weight of <1000 Dalton. The average molecular weights of the soy and yeast hydrolysates are preferably between about 220 and 375 daltons. The pH value of the soy hydrolysate and the yeast hydrolysate should be in the range between about 6.5 and 7.5. The total nitrogen content should be about between 8 and 11 %, preferably, between 9.0 and 10.0% and the ash content <18%. An advantageous hydrolysate is characterized by the feature that it has a free amino acids content of between about 5 and 30%. Endotoxin content, if any, should be <500 U/g.
[32] One medium according to the invention has the following constituency: synthetic minimal medium (DMEM/HAM's F12 (1 :1 ) medium (1-25 g/L), soy hydrolysate (0.5-10 g/L) and yeast hydrolysate (0.5-3g/L), L-glutamine (0.05-1 g/L), NaHC03 (0.1-10 g/L). The pH of the medium is between pH 6.8 and 7.6, preferably between pH 7.0 and 7.3.
[33] As is apparent to the skilled person, the term "about" in the context of numerical values and ranges refers to values or ranges that approximate or are close to the recited values or ranges such that the invention can perform as intended, such as having promoting a desired degree of cell growth, as is apparent from the teachings contained herein, and applies to all values. Thus, this term encompasses values beyond those resulting from systematic error.
[34] It has been surprisingly found that an animal protein free basal medium supplemented with yeast hydrolysate and soy hydrolysate within the range according to the present invention is more favorable for cell growth rate, cell metabolic activity and final cell density as compared to the medias described in the prior art. This was even more surprising in view of the teaching of WO 98/15614 showing that higher plant peptide concentrations are less optimal. With an animal protein free medium of the invention comprising yeast hydrolysate and soy hydrolysate as described herein, cells showed higher growth rate, higher final cell density of the biomass and increased metabolic activity (expressed in oxygen consumption in % per min) compared to medium either comprising soy hydrolysate or yeast hydrolysate alone, even if the final concentration of yeast hydrolysate or soy hydrolysate solely added to the medium is equivalent to the sum of the combined hydrolysate concentration. For example, a final concentration of about 0.4% (w/v) yeast hydrolysate in the medium alone had an inhibitory effect on cell growth and cell density. A medium comprising 0.4% (w/v) or higher concentrations of soy hydrolysate reached no higher cell density than a medium comprising 0.3% (w/v). However, a medium comprising a combination of soy hydrolysate and yeast hydrolysate in a final total hydrolysate concentration of 0.4% (w/v) showed a significant increase in cell metabolic activity, cell growth and final cell density. [35] In accordance with the invention, the sum of the amount of the soy and yeast hydrolysate in the medium should be between about 0.2 % (w/v) and about 0.6% (w/v) with a higher ratio of soy hydrolysate in the medium compared to yeast hydrolysate. An optimal ratio between soy and yeast hydrolysate should be about 3 : 1 (soy / yeast), respectively.
[36] The media of the invention as described herein is in particular useful to cultivate cells. Within the scope of the invention, the term "cells" means a generic term and encompass the cultivation of individual cells, tissues, organs, insect cells, avian cells, mammalian cells, primary cells, continuous cell lines, stem cells and/or genetically engineered cells, such as recombinant cells expressing a hetereologous polypeptide or protein. Recombinant cells include, for example, CHO cells or BHK cells expressing heterologous polypeptides or proteins, such as a growth factor or a blood factor. Cells often used for the propagation of virus include VERO cells and CV-1 cells.
[37] Mammalian cells suitable for cultivation in the cell culture medium of the present invention include those of human origin, which may be primary cells derived from a tissue sample, diploid cell strains, transformed cells or established cell lines. Mammalian cells can include human and non-human cells alike. Mammalian cells of non-human origin can be monkey kidney cells, bovine kidney cells, dog kidney cells, pig kidney cells, rabbit kidney cells, mouse kidney cells, rat kidney cells, sheep kidney cells, hamster kidney cells, Chinese hamster ovarian cells or an animal cell derived from any tissue. In particular, mammalian cells that can cultivated in the culture medium can be BSC-1 cells, LLC-MK cells, CV-1 cells, COS- cells, COS-1 cells, COS-3 cells, COS-7 cells, VERO cells, MDBK cells, MDCK cells, CRFK cells, RAF cells, RK-cells, TCMK-1 cells, LLC-PK cells, PK15 cells, LLC-RK cells, MDOK cells, BHK-21 cells, CHO cells, 293 cells, NS-1 cells MRC-5 cells, Wl- 38 cells, BHK cells, 293 cells and RK-cells. Examples or recombinant cells include CHO cells expressing Factor VIII, Fll, FIX, FX, vWF, for example, all of which are known to the person skilled in the art.
[38] The terms "continuous cells" or "continuous cell line" (CCL), in their various grammatical forms, mean cultured cells that replicate indefinitely and are capable of growing in suspension culture or large scale cultivation in bioreactor. The unrestricted growth of CCLs permits long-term cultivation from a standardized cell substrate and low costs. Mammalian cell lines can be selected from the group of CHO cells, COS cells, VERO cells, LLK-MK2 cells, NS-1 cells, MDBK cells, MDCK cells, MRC-5 cells, WI-38 cells, BHK cells, CV-1 cells, rabbit kidney (RK) cells and other cell lines as disclosed by Butler et al. BIOS Scientific Publisher p.1-24 (1992), which is incorporated herein by reference. The CCLs are preferably tested for absence of adventitious agents, such as bacteria, fungi, mycoplasma, protozoans and viruses.
[39] The term "cell culture," in its various grammatical forms, refers to cells grown in suspension, roller bottles, flasks and the like. Large scale approaches, such as bioreactors, including adherent cells growing attached to microcarriers in stirred fermentors, also are included. Moreover, it is possible to not only to culture surface-dependent cells, but also to use the suspension culture techniques with the inventive media. If the cells are grown on microcarriers, the microcarrier can be selected from the group of microcarriers based on dextran, collagen, plastic, gelatin and cellulose and others as described in Butler, Spier & Griffiths, Animal cell Biotechnology 3:283-303 (1988). Porous carriers, such as e.g. Cytoline® or Cytopore®, as well as dextran-based carriers, such as DEAE-dextran (Cytodex 1®), quaternary amine-coated dextran (Cytodex 2®) or gelatin-based carriers, such as gelatin-coated dextran (Cytodex 3®) are suitable. These carriers can be obtained from Pharmacia.
[40] The cells are preferably grown from the ampoule to the biomass in the animal protein free media and kept under culture medium conditions during cell culture growth and product production process. Cells that have already been adapted to the media are preferably used. It has been found that not only increased yields be achieved with such pre-adapted cells, but their stability for cultivation also is clearly improved by the use of the medium in accordance with the invention.
[41] The term "cultivation," in its various grammatical forms, refers to the maintenance of the cells in vitro under conditions permissive for growth and continued viability. Mammalian cells are typically cultivated in a cell incubator at about 37°C, with the culture medium having an optimal pH in the range of about 6.8 to 7.6, preferably between 7.0 and 7.3. Cells in batch culture might have a complete medium change about every 2 to 3 days, or more or less frequently, if required. Cells in perfusion culture (e.g. in bioreactor or fermenter) might have a fresh media change on a continuously recirculating basis. Cultivation approaches can include, depending on context and need, the sub-cultivation, passaging and propagation of the cells.
[42] The invention thus provides methods for cultivating cells comprising the steps of growing cells in a basal medium comprising yeast hydrolysate and soy hydrolysate. Preferably the cells are grown in a medium comprising soy hydrolysate in a concentration of 0.05% (w/v) to 1.0 % (w/v) and yeast hydrolysate in a concentration of 0.05% (w/v) to 0.3 % (w/v). According to this aspect of the invention, the cells are grown from small scale to large scale biomass in animal protein free media of the invention. The passaging and subcultivation of the cells to obtain a cell culture biomass is preferably performed with a non-animal-derived protease, such as Pronase or a purified fraction thereof. One protease is the purified trypsin-like fraction of Streptomyces griseus (SGT) as described in U.S. application serial no. 10/006,223, the entirety of which is hereby incorporated by reference. To avoid animal-derived material during cultivation of a cell culture, in particular during cultivation of adherent cells that grow attached to a carrier, the carrier is preferably a synthetic carrier, or a microcarrier coated with a non-animal derived material. For example a DEAE-dextran or quaternary amine-coated dextran microcarrier.
[43] The invention also provides an animal protein free cell culture process, wherein cells are cultivated, sub-cultivated and passaged under conditions devoid of animal proteins. The process comprising the steps of providing an animal protein free medium comprising yeast hydrolysate and soy hydrolysate, growing cells in said medium, passaging and sub-cultivating said cells grown in that medium using a non- animal-derived protease, further growing the sub-cultivated cells to reach a confluent cell density and repeating the steps of sub-cultivation and growth of cells until the final cell culture biomass desired is reached. The process includes the growth of the cells in animal protein free medium, sub-cultivating and passaging the cells using a non-animal derived protease, preferably a purified trypsin-like fraction of Streptomyces griseus (SGT). During cultivation of adherent cells that grow attached to a carrier, the carrier is preferably a synthetic carrier, or a microcarrier coated with a non-animal derived material. By the combination of these steps the use of animal proteins can be avoided.
[44] Cells suitable for growth in the animal protein free media of the present invention include, but are not limited to, BSC-1 cells, LLC-MK cells, CV-1 cells, VERO cells, MDBK cells, MDCK cells, CRFK cells, RAF cells, TCMK-1 cells, LLC- PK cells, PK15 cells, LLC-RK cells, MDOK cells, RK-cells, BHK-21 cells, WI-38 cells, 293 cells and/or MRC-5 cells. These cells can be infected with viruses, such as an orthomyxovirus, paramyxovirus, reovirus, picornavirus, flavivirus, arenavirus, herpesvirus, poxvirus, coronavirus, adenovirus and other viruses known to the skilled person. More specifically, the virus used to infect the cell culture can be Influenza virus, vaccinia virus and variola, fowlpox virus, cowpox virus, tick-borne encephalitis virus (TBE), poliovirus, Hepatitis A Virus, Ross River Virus, Yellow fever virus and a chimeric virus derived thereof, West nile virus, Japanese encephalitis virus, rubella virus, hepatitis C virus (HCV), mumps virus, measles virus, respiratory syncytial virus (RSV), herpes simplex virus (HSV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), rotavirus, foot and mouth disease virus (FMDV). It is within the knowledge of the skilled in the art to select the virus and the cells for which the virus is can be propagated. The cells can be cultivated in the media of the invention and grown to reach an optimal cell density prior infection with the respective virus. Surprisingly, a cell culture grown and propagated in an animal protein free medium of the invention shows a significant increase of virus yield productivity. Examples of different viruses propagated on cells cultivated and grown of the medium of the invention have shown a 2 to 5 fold increase of virus yield compared to a medium comprising solely yeast extract. This makes the system more favorable for cell growth and virus production processes than the those described in the prior art.
[45] According to an embodiment of the invention, the cells are VERO cells and the virus is selected from the group of Influenza Virus, TBE-Virus, vaccinia virus, poliovirus, Hepatitis A Virus, Ross River Virus, Yellow fever virus and a chimeric virus derived thereof, West nile virus, Japanese encephalitis virus, rubella virus, HCV, mumps virus, measles virus, respiratory syncytial virus, HSV, CMV, EBV, rotavirus. Other viruses known to grow in VERO cells also can be used. [46] The invention also provides for production of vaccinia virus by providing a culture of cells grown and cultivated in an animal protein free medium comprising yeast hydrolysate and soy hydrolysate, infecting said cells with a vaccinia virus and incubating the culture of cells to propagate the vaccinia virus. Preferably, the cells are grown in a medium comprising soy hydrolysate in a concentration of about 0.05% (w/v) to about 1.0% (w/v) and yeast hydrolysate concentration of about 0.05% (w/v) to about 0.3% (w/v). According to this aspect of the invention the cells can VERO cells, CV-1 cells, RK-cells, BHK-21 cells, MRC-5 cells, or any cell to which vaccinia virus can be grown. The vaccinia virus can be a naturally-occurring vaccinia virus, smallpox virus vaccine strain, virulent vaccinia strains, attenuated vaccinia strains and a recombinant vaccinia viruses.
[47] Also provided is production of orthomyxovirus by providing a culture of cells cultivated and grown in an animal protein free medium made from a basal medium comprising yeast hydrolysate and soy hydrolysate, infecting the cells with a orthomyxovirus and incubating the culture of cells to propagate the orthomyxovirus. Preferably, the cells are grown in a medium comprising soy hydrolysate in a concentration of about 0.05% (w/v) to about 1.0 % (w/v) and yeast hydrolysate in a concentration of about 0.05% (w/v) to about 0.3 % (w/v). The cells can be BSC-1 cells, CV-1 cells, VERO cells, MDBK cells, MDCK cells, MDOK cells, BHK-21 cells, WI-38 cells, MRC-5 cells or any cell to which orthomyxovirus can be propagated. The orthomyxovirus can be a Influenza virus, such as Influenza A, B or C.
[48] There also is provided production of Ross River Virus by providing a culture of cells grown and cultivated in an animal protein free medium made from a basal medium comprising yeast hydrolysate and soy hydrolysate, infecting said cells with a Ross River Virus and incubating the culture of cells to propagate the Ross River Virus. Preferably, the cells are grown in a medium comprising soy hydrolysate at a concentration of about 0.05% (w/v) to about 1.0 % (w/v) and yeast hydrolysate at a concentration of about 0.05% (w/v) to about 0.3 % (w/v). The cells can be BSC- 1 cells, CV-1 cells, VERO cells, MDBK cells, MDCK cells, CRFK cells, BHK-21 cells, WI-38 cells, MRC-5 cells or any cell to which Ross River Virus can be propagated.
[49] Additionally, there is provided production of Flavivirus by providing a culture of cells grown and cultivated on an animal protein free medium made from a basal medium comprising yeast hydrolysate and soy hydrolysate, infecting the cells with a Flavivirus and incubating the culture of cells to propagate the Flavivirus. Preferably, the cells are grown in a medium comprising soy hydrolysate in a concentration of about 0.05% (w/v) to about 1.0 % (w/v) and yeast hydrolysate in a concentration of about 0.05% (w/v) to about 0.3 % (w/v). The Flavivirus can be a Yellow fever virus, or a recombinant of chimeric derivatives thereof, Japanese encephalitis virus, Tick-borne encephalitis virus, West nile Virus, and Hepatitis C virus. The cells types identified herein can be used for the propagation of flavivirus.
[50] There is provided production of Picornavirus by providing a culture of cells grown and cultivated on an animal protein free medium made from a basal medium comprising yeast hydrolysate and soy hydrolysate, infecting the cells with a Picornavirus and incubating the culture of cells to propagate the Picornavirus. Preferably, the cells are grown in a medium comprising soy hydrolysate in a concentration of about 0.05% (w/v) to about 1.0 % (w/v) and yeast hydrolysate in a concentration of about 0.05% (w/v) to about 0.3 % (w/v). The Picornavirus can be a poliovirus or hepatitis A virus. The cells types identified herein can be used for the propagation of Picornavirus.
[51] The invention also provides methods of producing immunogenic compositions comprising a virus or a virus antigen comprising the steps of providing a culture of animal cells, wherein the cells are selected from the group of monkey kidney cells, bovine kidney cells, dog kidney cells, pig kidney cells, mouse kidney cells, rat kidney cells, sheep kidney cells, rabbit kidney cells, hamster kidney cells and human cells that have been grown the a medium of the invention; infecting the cells with a virus selected from the group of orthomyxoviruses, paramyxoviruses, reoviruses, picornaviruses, flaviviruses, arenaviruses, herpesviruses, poxviruses, coronaviruses and adenoviruses, incubating the culture of cells to propagate the virus, harvesting the virus produced and preparing an immunogenic composition from the virus harvested. The virus produced and harvested can be purified with a method known in the art, such as ion exchange or gel filtration.
[52] Having now generally described this invention, the same will be further understood by reference to the following examples which are provided herein for purposes of illustration and are not limiting in any manner. EXAMPLES:
EXAMPLE 1
Formulation of the culture medium
[53] Animal protein free medium is prepared with a basal DMEM/ HAM's F12 (1 :1 ) medium that is supplemented with inorganic salts, amino acids, vitamins and other components. Also added are sodium bicarbonate (1-3 g/ L), L-Glutamine (0.1 to 1 g /L) and varying concentrations of soy hydrolysate (Quest Technologies, New York) or yeast hydrolysate (Deutsche Hefewerke, Germany) or combinations thereof. EXAMPLE 2
Propagation of cells in animal protein free medium VERO cells in animal protein free medium
[54] VERO cells (African Green Monkey, Cercopthecus aethiops, kidney) were used as cell line. The cells have been obtained from the American Type Cell Culture Collection, Rockville, Maryland at a passage number 124 under the designation ATCC CCL 81. The cells were grown in various media as described herein.
[55] Cells of the working cell bank were expanded in T-flasks and roller bottles and microcarrier systems with a split ratio 1 :6 -1 :8. The cells were grown at 37°C for 6-8 days. The culture conditions of oxygen saturation 20%+/- 10% and pH 7.1 +/- 0.35 were kept constant. At the end of biomass production when cell have reached confluence growth, the cell density and oxygen consumption rate was determined.
[56] The cell number of the biomass of the cell culture at the end of biomass production was determined either by trypsinization of the cells and counting with a CASY® cell counter (method A) as described by Scharfe et al. Biotechnologie in LaborPraxis 10:1096-1103 1988) or by citric acid and crystal violet treatment followed by counting with a haemocytometer (method B) as described by Sanford et al., J. Nat'l Cancer Inst. 11 :773-795 (1951). [57] VERO cells were cultivated and grown in animal protein free medium comprising yeast hydrolysate in a concentration of 0.05%, 0.1 %, 0.2%, 0.3% or 0.4%, 0.5% (w/v), or soy hydrolysate in a concentration of 0.05%, 0.1 %, 0.2%, 0.3% or 0.4%, 0.5% (w/v), or soy hydrolysate and yeast hydrolysate in a concentration of yeast to soy (yeast / soy) of 0.05% / 0.05% (w/v), 0.1 % / 0.2% (w/v), 0.1 % / 0.3% (w/v), 0.2% / 0.2% (w/v), 0.3% / 0.2% (w/v) or 0.2% / 1.0% (w/v). The cell density of the cell culture at the end of biomass production in animal protein free medium comprising varying concentrations of soy hydrolysate, yeast hydrolysate or combinations thereof was calculated by methods A and B.
[58] The results demonstrate that yeast and soy hydrolysate alone supported cell growth. At a concentration of 0.1 % yeast hydrolysate a cell density of about 11.8 x 105 cells/ ml was reached, however increasing concentration of yeast hydrolysate to higher than 0.3% (w/v) had a negative effect on cell growth and consequently on cell density. Concentrations of soy hydrolysate alone between 0.1 % (w/v) to 0.2% (w/v) showed less cell growth and cell density than with soy concentrations of 0.3% (w/v) and 0.4% (w/v). Nevertheless, cell density and oxygen consumption of cells cultivated in medium comprising 0.3% (w/v) or 0.4% (w/v) soy hydrolysate did not differ significantly and higher concentrations to about 1% w/v of soy hydrolysate had no positive effect on cell growth. The optimal concentration of soy hydrolysate alone was determined to be between 0.2% w/v to 1.0% w/v. By supplementation of the basal medium with a combination of soy hydrolysate and yeast hydrolysate the final cell density reached was significantly increased compared to a medium comprising solely soy or yeast hydrolysate. The cell density reached at a concentration of 0.05% (w/v) soy and 0.05% (w/v) yeast was about 12.1 x 105 cells /ml and had a higher cell culture cell density compared to cells grown in medium comprising solely either 0.1 % (w/v) soy hydrolysate (10 x 105 cells/ml) or 0.1% (w/v) yeast hydrolysate (11.8 x 105 cells/ml). The cell density of a cell culture grown in a medium comprising yeast with a concentration of 0.2% (w/v) and soy of 1.0% (w/v) was similar to the density obtained in medium comprising 0.05% (w/v) soy and 0.05% yeast (w/v).
[59] The most significant effect on cell growth was in medium wherein the soy hydrolysate concentration compared to yeast hydrolysate was about 2-3 times higher. Cells grown in a medium comprising soy hydrolysate at concentration of about 0.3% (w/v) and yeast hydrolysate of about 0.1% (w/v) reached a cells density of about 21.0 x 105 cells /ml and showed therefore an approximately 2 times higher cell density compared to cells grown solely in soy hydrolysate of about 0.4% (w/v) and an about 2.5 times higher cells density compared to cells cultivated in medium comprising solely yeast hydrolysate of about 0.4 % (w/v). The metabolic activity of cells cultivated in medium comprising yeast and soy hydrolysate also was higher compared to cells grown in medium solely comprising yeast or soy hydrolysate. The oxygen consumption rate was 1.5 (% per min.) in a medium comprising 0.1 % (w/v) yeast hydrolysate and less than 1.0 (% per min.) in medium comprising 0.4% (w/v) yeast hydrolysate or soy hydrolysate alone. In a medium comprising 0.1 % (w/v) yeast hydrolysate and 0.3% (w/v) soy hydrolysate the oxygen consumption was about 2.9 (% per min.), which was about 2 times higher than of cells cultivated in a medium comprising solely soy or yeast hydrolysate.
[60] Additionally the cell cycle, which is 7 days in animal protein free medium supplemented with yeast or soy hydrolysate alone, is reduced to 6 days in the hydrolysate combination (soy and yeast) medium.
EXAMPLE 3
Propagation of recombinant cells
[61] A cell culture of recombinant mammalian cells, such as rFVIII-CHO cells, are cultivated in a 10L stirred tank with perfusion. A medium in accordance with Example 1 is used as cultivation and growth medium. The cells are immobilized on a porous microcarrier (Cytopore®, Pharmacia) and cultivated for at least 6 weeks. The perfusion rate is 4 volume changes per day; the pH is 6.9 to 7.2; the 02 concentration is approximately 20-50% and the temperature is 37°C. The cell density is determined.
EXAMPLE 4
Comparison of virus antigen production on VERO cells grown in medium supplemented with yeast hydrolysate and soy hydrolysate a. Production of cell culture biomass [62] VERO cells with a defined passage number were thawed from liquid nitrogen and passaged in roux and roller bottles to produce sufficient cells to inoculate a 1.5 liter bioreactor. Cells are either grown in basal medium supplemented either with yeast hydrolysate or with a combination of yeast hydrolysate and soy hydrolysate as described in Examples 1 and 2. After reaching confluency with a final cell density of 1.5 x 106 cells / ml, the cells were released from the microcarrier with a purified fraction of Pronase, the S. griseus trypsin (SGT) as described in U.S. application serial no. 10/006,223 and transferred to a 10 liter bioreactor. This in turn was used as an inoculum for a 100 liter bioreactor having a microcarrier concentration of 3.0 g/l. Starting from a working cell bank ampoule containing 107 cells, about 30 generations were needed to reach the final confluent VERO cell biomass in the last fermenter vessel. The cells were grown at 37°C. The culture conditions of oxygen saturation 20% +/- 10% and pH 7.1 +/- 0.35 were kept constant during virus propagation process.
[63] Cells of the working cell bank of VERO cells were expanded in T-flasks and roller bottles with a split ratio of 1 :6. Further propagation of the cells was performed in 1.5, 10 and a 50 I stirred fermenter as bioreactor using Cytodexl® microcarrier as attachment substrate. The cells were grown at 37°C. The culture conditions of oxygen saturation 20% +/- 10% and pH 7.1 +/- 0.35 were kept constant during virus propagation process.
b. Propagation of Influenza Virus
[64] VERO cells were infected with two different influenza strains, New Caledonia A/H1 N1 and Panama A/H3N2, and propagated in the respective medium. At the end of the virus propagation process the clarified supernatant containing the virus was purified by ultracentrifugation. The harvests of the VERO cells culture with either solely yeast hydrolysate or with yeast and soy hydrolysate were compared on the basis of the volumetric antigen productivity (total SRD, single radial immunodiffusion) and the antigen content of the supernatant at the end of the run (density gradient purified antigen). The yields for both media formulations were compared and are summarized in Table 1. [65] Table 1. Comparison of product yield from VERO Influenza production in different media composition
Figure imgf000022_0001
The combination of yeast hydrolysate and soy hydrolysate shows a marked improvement over yeast hydrolysate alone.
c. Production of Poxvirus
[66] VERO cells were infected with a smallpox vaccine production strain (Dryvax, Wyeth Vaccines, obtained from Acambis, Inc., a calf-lymph vaccine strain adapted for growth in permanent cell line) adapted to growth in animal protein free VERO cells by serial passaging at a multiplicity of infection (m.o.i.) of 0.1 - 0.3. After an incubation time of 2-4 days at 37°C the cells were harvested and virus was recovered from the cells.
[67] Table 2 shows the results of virus yield obtained of cells grown in basal medium supplemented with yeast hydrolysate alone or with yeast and soy hydrolysate. [68] TABLE 2: Determination of Vaccinia virus titer at the end of the production cycle in the bioreactor system.
Figure imgf000023_0001
The combination of yeast hydrolysate and soy hydrolysate shows a marked improvement over yeast hydrolysate alone.
d. Production of Ross River Virus
[69] VERO cell culture obtained as described herein were infected with Ross River Virus at a multiplicity of infection (m.o.i.) of 0.1 - 0.3. After an incubation time of 2-4 days at 37°C, the cells were harvested and virus was recovered from the cells. Table 3 shows the results of virus yield obtained of cells grown in basal medium supplemented with yeast hydrolysate alone or with yeast and soy hydrolysate.
[70] TABLE 3 Determination of Ross River Virus titer at the end of the production cycle in the bioreactor system.
Figure imgf000023_0002
The combination of yeast hydrolysate and soy hydrolysate shows a marked improvement over yeast hydrolysate alone.
[71] It is to be understood that the description, specific examples and data, while indicating exemplary embodiments, are given by way of illustration and are not intended to limit the present invention. Various changes and modifications within the present invention will become apparent to the skilled artisan from the discussion, disclosure and data contained herein, and thus are considered part of the invention.

Claims

What is claimed is:
1. An animal protein free cell culture medium comprising soy hydrolysate and yeast hydrolysate.
2. The animal protein free cell culture medium according to claim 1 , wherein the soy hydrolysate is present in a concentration of at least 0.05% (w/v) and yeast hydrolysate is present in a concentration of at least 0.05% (w/v).
3. The animal protein free cell culture medium according to claim 1 , wherein the soy hydrolysate is present in a concentration of less than 1.0% (w/v) and yeast hydrolysate is present in a concentration of less than 0.3% (w/v).
4. The animal protein free cell culture medium according to claim 1 , wherein the soy hydrolysate is present in a concentration of between about 0.2% (w/v) to about 0.6% (w/v) and yeast hydrolysate is present in a concentration of between about 0.05% (w/v) to about 0.2 % (w/v).
5. The animal protein free cell culture medium according to claim 1 , wherein the soy hydrolysate is present in a concentration of between about 0.25% (w/v) to about 0.35% (w/v) and the yeast hydrolysate is present in a concentration of between about 0.05% (w/v) to about 0.15% (w/v).
6. The animal protein free cell culture medium according to claim 1 , wherein the soy hydrolysate is present in a concentration of about 0.3% (w/v) and the yeast hydrolysate is present in a concentration of about 0.1 % (w/v).
7. The animal protein free cell culture medium according to claim 1 , wherein 3 parts by weight soy hydrolysate are present to 1 part by weight yeast hydrolysate.
8. The animal protein free cell culture medium according to claim 1, wherein the yeast hydrolysate is a ultrafiltrated purified yeast hydrolysate, and wherein at least 40% of said yeast hydrolysate has a molecular weight of less than or equal to 500 Daltons.
9. The animal protein free cell culture medium according to claim 1 , wherein the soy hydrolysate is a ultrafiltrated purified soy hydrolysate, and wherein at least 40% of said soy hydrolysate has a molecular weight of less than or equal to 500 Daltons.
10. A method of producing an animal protein free cell culture medium, wherein a basal medium that is free of any animal proteins is supplemented with a yeast hydrolysate and a soy hydrolysate.
11. The method according to claim 10, wherein the concentration of soy hydrolysate is at least 0. 05% (w/v) and the concentration of yeast hydrolysate is at least 0.05% (w/v).
12. The method according to claim 10, wherein the concentration of soy hydrolysate is less than 1.0 % (w/v) and the concentration of yeast hydrolysate is less than 0.3 % (w/v).
13. A method of cultivating a cell culture of cells comprising: providing a medium comprising soy hydrolysate at a concentration of about 0.05% (w/v) to about 1 % (w/v) and yeast hydrolysate at a concentration of about 0.05% (w/v) to about 0.3% (w/v); and propagating the cells in the medium to form the cell culture.
14. The method according to claim 13, wherein the cells are animal cells selected from the group consisting of insect cells, avian cells and mammalian cells.
15. The method according to claim 13, wherein the cells are recombinant cells.
16. The method according to claim 13, wherein the cells are selected from the group of cells consisting of BSC-1 cells, LLC-MK cells, CV-1 cells, COS-cells, VERO cells, MDBK cells, MDCK cells, CRFK cells, RAF cells, RK-cells, TCMK-1 cells, LLC- PK cells, PK15 cells, LLC-RK cells, MDOK cells, BHK-21 cells, CHO cells, NS-1 cells MRC-5 cells, WI-38 cells, BHK cells, and RK-cells.
17. An animal protein free confluent cell culture process, comprising: providing an animal protein free medium comprising soy hydrolysate and yeast hydrolysate; growing the cells in the medium, and passaging and sub-cultivating the cells grown in the medium while in contact with a non-animal-derived protease in order to obtain a confluent cell culture.
18. A culture of cells cultivated in an animal protein free medium, wherein the medium comprises soy hydrolysate at a concentration of about 0. 05% (w/v) to about 1 % (w/v) and yeast hydrolysate at a concentration of about 0.05% (w/v) to about 0.3% (w/v).
19. The culture of cells according to claim 18, wherein the cells are animal cells selected from the group consisting of insect cells, avian cells and mammalian cells.
20. The culture of cells according to claim 18, wherein the cells are recombinant cells.
21. The culture of cells cultivated according to claim 18, wherein the cells are infected with a virus.
22. The culture of cells according to claim 18, wherein the cells are selected from the group of cells consisting of BSC-1 cells, LLC-MK cells, CV-1 cells, COS-cells, VERO cells, MDBK cells, MDCK cells, CRFK cells, RAF cells, RK-cells, TCMK-1 cells, LLC-PK cells, PK15 cells, LLC-RK cells, MDOK cells, BHK-21 cells, CHO cells, NS-1 cells MRC-5 cells, WI-38 cells, BHK cells, and RK-cells.
23. A method for producing a virus, comprising: providing a culture of cells that have been grown in an animal protein free medium comprising soy hydrolysate at a concentration of about 0.05% (w/v) to about 1 % (w/v) and yeast hydrolysate at a concentration of about 0.05% (w/v) to about 0.3% (w/v); infecting the cells with a virus; and incubating the infected cells to propagate the virus.
24. The method according to claim 21 , wherein the cells are animal cells selected from the group consisting of insect cells, avian cells and mammalian cells.
25. A method for producing vaccinia virus, comprising: providing a culture of cells that have been grown in an animal protein free medium comprising soy hydrolysate at a concentration of about 0.05% (w/v) to about 1 % (w/v) and yeast hydrolysate at a concentration of about 0.05% (w/v) to about 0.3% (w/v); infecting the cells with vaccinia virus; and incubating the infected cells to propagate vaccinia virus.
26. A method for producing coronavirus, comprising: providing a culture of cells that have been grown in an animal protein free medium comprising soy hydrolysate at a concentration of about 0.05% (w/v) to about 1 % (w/v) and yeast hydrolysate at a concentration of about 0.05% (w/v) to about 0.3% (w/v); infecting the cells with coronavirus; and incubating the infected cells to propagate coronavirus.
27. A method for producing orthomyxovirus, comprising: providing a culture of cells that have been grown in an animal protein free medium comprising soy hydrolysate at a concentration of about 0.05% (w/v) to about 1% (w/v) and yeast hydrolysate at a concentration of about 0.05% (w/v) to about 0.3% (w/v); infecting the cells with orthomyxovirus; and incubating the infected cells to propagate orthomyxovirus.
28. The method according to claim 27, wherein the orthomyxovirus is Influenza A, B or C Virus.
29. A method for producing Ross River virus, comprising providing a culture of cells that have been grown in an animal protein free medium comprising soy hydrolysate at a concentration of about 0.05% (w/v) to about 1 % (w/v) and yeast hydrolysate at a concentration of about 0.05% (w/v) to about 0.3% (w/v); infecting the cells with Ross River virus; and incubating the infected cells to propagate Ross River virus.
30. A method for producing Flavivirus, comprising: providing a culture of cells that have been grown in an animal protein free medium comprising soy hydrolysate at a concentration of about 0.05% (w/v) to about 1 % (w/v) and yeast hydrolysate at a concentration of about 0.05% (w/v) to about 0.3% (w/v); infecting the cells with Flavivirus; and incubating the infected cells to propagate Flavivirus.
31. The method according to claim 30, wherein the Flavivirus is selected from the group consisting of Yellow fever virus, Japanese encephalitis virus, Tick-borne encephalitis virus, West nile Virus and Hepatitis C virus.
32. A method for producing picornavirus, comprising: providing a culture of cells that have been grown in an animal protein free medium comprising soy hydrolysate at a concentration of about 0.05% (w/v) to about 1 % (w/v) and yeast hydrolysate at a concentration of about 0.05% (w/v) to about 0.3% (w/v); infecting the cells with picornavirus; and incubating the infected cells to propagate picornavirus.
33. The method according to claim 32, wherein the picornavirus is selected from the group consisting of poliovirus and hepatitis A virus.
34. A method of producing an immunogenic composition comprising a virus or a virus antigen, wherein the method comprises: providing a culture of cells that have been grown in an animal protein free medium comprising soy hydrolysate at a concentration of about 0.05% (w/v) to about 1 % (w/v) and yeast hydrolysate at a concentration of about 0.05% (w/v) to about 0.3% (w/v); infecting the cells with the virus; incubating the infected cells to propagate the virus; harvesting the virus or virus antigen produced prepare an immunogenic composition from the harvested virus or virus antigen.
35. The method according to claim 34, wherein said harvested virus or virus antigen is subjected to purification.
36. A method of producing an immunogenic composition comprising a virus or a virus antigen, wherein the method comprises: providing a culture of mammalian cells, wherein the cells are selected from the group of monkey kidney cells, bovine kidney cells, dog kidney cells, pig kidney cells, mouse kidney cells, rat kidney cells, sheep kidney cells, hamster kidney cells and human cells that have been grown in an animal protein free culture medium comprising a soy hydrolysate and a yeast hydrolysate; infecting the cells with a virus selected from the group of orthomyxoviruses, paramyxoviruses, reoviruses, picornaviruses, flaviviruses, arenaviruses, herpesviruses, poxviruses, coronaviruses and adenoviruses; incubating the culture of cells to propagate the virus; harvesting the virus or virus antigen so produced; and preparing an immunogenic composition from the harvested virus or virus antigen.
37. A culture of cells infected with orthomyxovirus, wherein the cells are cultivated in an animal protein free medium, wherein the medium comprises soy hydrolysate and yeast hydrolysate.
38. The culture according to claim 37, wherein the soy hydrolysate is at a concentration of about 0. 05% (w/v) to about 1% (w/v) and yeast hydrolysate at a concentration of about 0.05% (w/v) to about 0.3% (w/v).
39. A culture of cells infected with poxvirus, wherein the cells are cultivated in an animal protein free medium, wherein the medium comprises soy hydrolysate and yeast hydrolysate.
40. The culture according to claim 39, wherein the soy hydrolysate is at a concentration of about 0. 05% (w/v) to about 1% (w/v) and yeast hydrolysate at a concentration of about 0.05% (w/v) to about 0.3% (w/v).
41. A culture of cells infected with herpesvirus, wherein the cells are cultivated in an animal protein free medium, wherein the medium comprises soy hydrolysate and yeast hydrolysate.
42. The culture according to claim 41 , wherein the soy hydrolysate is at a concentration of about 0. 05% (w/v) to about 1% (w/v) and yeast hydrolysate at a concentration of about 0.05% (w/v) to about 0.3% (w/v).
43. An orthomyxovirus preparation that is free of animal proteins, wherein the preparation is obtainable by cultivating cells infected with influenza virus in an animal protein free medium, wherein the medium comprises soy hydrolysate and yeast hydrolysate.
44. A herpes virus preparation that is free of animal proteins, wherein the preparation is obtainable by cultivating cells infected with influenza virus in an animal protein free medium, wherein the medium comprises soy hydrolysate and yeast hydrolysate.
45. A poxvirus preparation that is free of animal proteins, wherein the preparation is obtainable by cultivating cells infected with influenza virus in an animal protein free medium, wherein the medium comprises soy hydrolysate and yeast hydrolysate.
PCT/EP2003/007341 2002-07-09 2003-07-08 Animal protein free media for cultivation of cells WO2004005493A1 (en)

Priority Applications (10)

Application Number Priority Date Filing Date Title
EP20030762657 EP1520008B1 (en) 2002-07-09 2003-07-08 Animal protein free media for cultivation of cells
CA 2491992 CA2491992A1 (en) 2002-07-09 2003-07-08 Animal protein free media for cultivation of cells
JP2004518745A JP2005532057A (en) 2002-07-09 2003-07-08 Animal protein-free medium for culturing cells
MXPA05000418A MXPA05000418A (en) 2002-07-09 2003-07-08 Animal protein free media for cultivation of cells.
AU2003249990A AU2003249990B2 (en) 2002-07-09 2003-07-08 Animal protein free media for cultivation of cells
SI200332208T SI1520008T1 (en) 2002-07-09 2003-07-08 Animal protein free media for cultivation of cells
ES03762657T ES2394085T3 (en) 2002-07-09 2003-07-08 Animal protein free medium for cell culture
NZ538094A NZ538094A (en) 2002-07-09 2003-07-08 Serum free and animal protein free culture medium for cultivation of cells
DK03762657T DK1520008T3 (en) 2002-07-09 2003-07-08 Medium free of animal protein for cell culture
HK06103671A HK1083635A1 (en) 2002-07-09 2006-03-23 Animal protein free media for cultivation of cells

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US39424302P 2002-07-09 2002-07-09
US60/394,243 2002-07-09

Publications (1)

Publication Number Publication Date
WO2004005493A1 true WO2004005493A1 (en) 2004-01-15

Family

ID=30115698

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2003/007341 WO2004005493A1 (en) 2002-07-09 2003-07-08 Animal protein free media for cultivation of cells

Country Status (15)

Country Link
US (3) US7955833B2 (en)
EP (2) EP2287288B1 (en)
JP (5) JP2005532057A (en)
CN (2) CN100591759C (en)
AU (1) AU2003249990B2 (en)
CA (1) CA2491992A1 (en)
DK (2) DK1520008T3 (en)
ES (2) ES2398706T3 (en)
HK (2) HK1083635A1 (en)
MX (1) MXPA05000418A (en)
NZ (1) NZ538094A (en)
PL (1) PL214284B1 (en)
PT (2) PT1520008E (en)
SI (1) SI1520008T1 (en)
WO (1) WO2004005493A1 (en)

Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005083058A1 (en) * 2004-03-01 2005-09-09 Ares Trading S.A. Use of a serum-free cell culture medium for the production of il-18bp in mammalian cells
WO2006045438A1 (en) * 2004-10-29 2006-05-04 Baxter International Inc. Animal protein-free media for cultivation of cells
EP1739167A1 (en) * 2004-04-19 2007-01-03 DENKA SEIKEN Co., Ltd. Method of producing virus
JP2007000077A (en) * 2005-06-23 2007-01-11 Hitachi Medical Corp Method for serum-free culture of adherent animal cell and culture medium for serum-free culture of adherent animal cell
KR100679112B1 (en) 2004-11-17 2007-02-07 삼성정밀화학 주식회사 Method for Animal Cell Culture
JP2008512443A (en) * 2004-09-09 2008-04-24 カイロン ベーリング ゲーエムベーハー アンド カンパニー Reduce potential iatrogenic risk associated with influenza vaccines
JPWO2006095431A1 (en) * 2005-03-10 2008-08-14 共立製薬株式会社 Cell line that can be cultured without animal-derived components, method for producing the same, virus production method using the same, and vaccine production method
JP2008536499A (en) * 2005-04-13 2008-09-11 メリアル リミテッド Porcine circovirus production assay
EP2017333A1 (en) * 2006-04-28 2009-01-21 Kyoritsu Seiyaku Corporation Feline cell capable of being cultured without animal protein, and method for production of virus and method for production of vaccine using the feline cell
US7955833B2 (en) 2002-07-09 2011-06-07 Baxter International Inc. Animal protein free media for cultivation of cells
US8021881B2 (en) 1999-09-28 2011-09-20 Baxter Innovations Gmbh Medium for the protein-free and serum-free cultivation of cells
US8080414B2 (en) 1997-06-20 2011-12-20 Baxter Innovations Gmbh Recombinant cell clones having increased stability and methods of making and using the same
AU2012238257B2 (en) * 2004-10-29 2014-04-10 Takeda Pharmaceutical Company Limited Animal Protein-Free Media for Cultivation of Cells
WO2015101510A1 (en) * 2013-12-30 2015-07-09 Baxter Healthcare Sa A method of predicting a performance characteristic of a plant or yeast hydrolysate and its use
WO2015086598A3 (en) * 2013-12-09 2015-10-01 Vibalogics Gmbh Method of culturing vero cells
CN105154389A (en) * 2015-10-15 2015-12-16 南京三生生物技术有限公司 Low-serum protein-free culture medium suitable for PK-15 cell growth and preparation method of culture medium
CN105385661A (en) * 2014-09-03 2016-03-09 普莱柯生物工程股份有限公司 Porcine circovirus type 2 large-scale cultivation method and applications thereof
EP1974014B1 (en) 2006-01-04 2017-04-19 Baxalta Incorporated Oligopeptide-free cell culture media
EP3152317B1 (en) 2014-06-04 2019-01-02 Amgen Inc. Methods for harvesting mammalian cell cultures
EP4093848A4 (en) * 2020-01-24 2024-08-07 Air Protein Inc Microorganism-derived protein hydrolysates, and methods of preparation and use thereof

Families Citing this family (41)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7425437B2 (en) * 1999-11-26 2008-09-16 Crucell Holland B.V. Vaccines against West Nile Virus
FR2836924B1 (en) * 2002-03-08 2005-01-14 Vivalis AVIAN CELL LINES USEFUL FOR THE PRODUCTION OF INTEREST SUBSTANCES
DK2368975T3 (en) * 2004-12-23 2015-01-05 Medimmune Llc Non-tumorigenic MDCK cell line for the propagation of viruses
FR2884255B1 (en) 2005-04-11 2010-11-05 Vivalis USE OF EBX AVIATION STEM CELL LINES FOR THE PRODUCTION OF INFLUENZA VACCINE
EP1879921B1 (en) 2005-05-12 2011-04-27 Crucell Holland B.V. Host cell specific binding molecules capable of neutralizing viruses and uses thereof
AU2007245192B2 (en) 2006-03-31 2012-04-12 Wisconsin Alumni Research Foundation High titer recombinant influenza viruses for vaccines
HUE027099T2 (en) * 2006-06-06 2016-08-29 Crucell Holland Bv Human binding molecules having killing activity against staphylococci and uses thereof
MX2009001477A (en) * 2006-08-09 2009-02-18 Vivalis Method of production of transgenic avian using embryonic stem cells.
RU2487935C2 (en) * 2006-09-15 2013-07-20 Медиммун, Ллк. Cell lines of madin-derby canine kidney supporting virus growth to high tires and method to use these cells in bioreactor
TW200902708A (en) 2007-04-23 2009-01-16 Wyeth Corp Methods of protein production using anti-senescence compounds
EP1985305A1 (en) 2007-04-24 2008-10-29 Vivalis Duck embryonic derived stem cell lines for the production of viral vaccines
EP1995309A1 (en) * 2007-05-21 2008-11-26 Vivalis Recombinant protein production in avian EBx® cells
US9474798B2 (en) 2007-06-18 2016-10-25 Wisconsin Alumni Research Foundation Influenza M2 protein mutant viruses as live influenza attenuated vaccines
US20090042253A1 (en) * 2007-08-09 2009-02-12 Wyeth Use of perfusion to enhance production of fed-batch cell culture in bioreactors
KR101818411B1 (en) * 2007-12-27 2018-01-12 박스알타 인코퍼레이티드 Cell culture processes
DK2240578T3 (en) * 2007-12-31 2019-06-17 Baxalta GmbH TOTALLY ESSENTIAL DEPOSIT FREE RECOMBINANT FURIN AND METHODS OF MANUFACTURE
KR101220239B1 (en) * 2008-04-01 2013-01-09 바이오스펙트럼 주식회사 Composition for Promoting Stem Cell Proliferation Comprising Vegetable Peptone
TW201012930A (en) 2008-06-16 2010-04-01 Intervet Int Bv Method of replicating viruses in suspension
AU2009296701B2 (en) 2008-09-24 2015-02-19 Medimmune, Llc Methods for cultivating cells, propagating and purifying viruses
WO2011011660A2 (en) * 2009-07-23 2011-01-27 Xcellerex, Inc. Drain down and re-feed of microcarrier bioreactor
JP2013507990A (en) 2009-10-26 2013-03-07 ダブリュエーアールエフ−ウィスコンシン アラムナイ リサーチ ファウンデーション High-titer recombinant influenza virus with enhanced replication in Vero cells
US10130697B2 (en) 2010-03-23 2018-11-20 Wisconsin Alumni Research Foundation (Warf) Vaccines comprising mutant attenuated influenza viruses
CN101988047B (en) * 2010-05-13 2013-07-03 维亚生物科技(上海)有限公司 Insect cell serum-free medium with low cost
CN102533634A (en) * 2012-02-22 2012-07-04 江阴剑桥生物技术有限公司 Serum-free protein-free chemical culture medium for Chinese hamster ovary (CHO) cells
EP3022296B1 (en) 2013-07-15 2022-12-28 Wisconsin Alumni Research Foundation High titer recombinant influenza viruses with enhanced replication in mdck or vero cells or eggs
US10053671B2 (en) 2014-06-20 2018-08-21 Wisconsin Alumni Research Foundation (Warf) Mutations that confer genetic stability to additional genes in influenza viruses
US10633422B2 (en) 2015-06-01 2020-04-28 Wisconsin Alumni Research Foundation (Warf) Influenza virus replication by inhibiting microRNA lec7C binding to influenza viral cRNA and mRNA
WO2017007839A1 (en) 2015-07-06 2017-01-12 Wisconsin Alumni Research Foundation (Warf) Improved influenza virus replication for vaccine development
CA3014435C (en) 2016-02-19 2023-04-18 Wisconsin Alumni Research Foundation (Warf) Improved influenza b virus replication for vaccine development
ES2901134T3 (en) * 2017-03-09 2022-03-21 Evonik Operations Gmbh Culture medium comprising oligopeptides
CN107043740A (en) * 2017-05-10 2017-08-15 浙江美保龙生物技术有限公司 A kind of MDBK cell culture fluids and preparation method thereof, application method
CA3120129A1 (en) * 2018-11-21 2020-05-28 Western Oncolytics Ltd. Manufacture of virus
MX2021006115A (en) * 2019-01-17 2021-07-07 Boehringer Ingelheim Animal Health Usa Inc Serum-free medium for avian vaccine production and uses thereof.
US11241492B2 (en) 2019-01-23 2022-02-08 Wisconsin Alumni Research Foundation (Warf) Mutations that confer genetic stability to genes in influenza viruses
WO2020153619A1 (en) * 2019-01-25 2020-07-30 바이로큐어 주식회사 Virus production method using bhk-21 cells
WO2020163804A1 (en) 2019-02-08 2020-08-13 Wisconsin Alumni Research Foundation (Warf) Humanized cell line
US11390649B2 (en) 2019-05-01 2022-07-19 Wisconsin Alumni Research Foundation (Warf) Influenza virus replication for vaccine development
US11807872B2 (en) 2019-08-27 2023-11-07 Wisconsin Alumni Research Foundation (Warf) Recombinant influenza viruses with stabilized HA for replication in eggs
WO2022103252A1 (en) * 2020-11-12 2022-05-19 Imu Education Sdn Bhd Culture medium for bacteria
KR102581772B1 (en) * 2021-03-30 2023-09-22 강원대학교 산학협력단 A method of optimal cultivation for preparing cultured meat using plant and insect protein hydrolysate and application of the same
WO2023235132A1 (en) * 2022-06-03 2023-12-07 Bluerock Therapeutics Lp Cell delivery vehicle and methods of using the same

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998015614A1 (en) * 1996-10-10 1998-04-16 Life Technologies, Inc. Animal cell culture media comprising plant-derived nutrients
WO1999047648A2 (en) * 1998-03-13 1999-09-23 Aventis Pasteur Medium and method for viral propagation and growth
WO2000003000A2 (en) * 1998-07-10 2000-01-20 Chugai Seiyaku Kabushiki Kaisha Serum-free medium for culturing animal cells
JP2001211878A (en) * 2000-02-04 2001-08-07 National Federation Of Dairy Cooperative Associations Method of culturing yogurt starter lactobacillus in high concentration, and method of producing yogurt from the obtained high concentration culture solution

Family Cites Families (71)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AT165999B (en) 1947-06-26 1950-05-25 Delle Atel Const Electr Device for protecting three-phase motors against overcurrent
FR2196386A1 (en) 1972-08-17 1974-03-15 Cudennec Alain Culture media selection - for identification of unknown bacteria
US4500513A (en) * 1979-05-15 1985-02-19 Miles Laboratories, Inc. Influenza vaccine production in liquid cell culture
US4282315A (en) * 1979-09-13 1981-08-04 Corning Glass Works Preparation of enriched whole virus radioligand
US4443540A (en) * 1980-05-09 1984-04-17 University Of Illinois Foundation Protein hydrolysis
DK207980A (en) * 1980-05-13 1981-11-14 Novo Industri As PROCEDURE FOR THE PREPARATION OF A FOAM OR EMERGENCY FOUNDANT ON SOY PROTEIN BASIS
DE3122669A1 (en) * 1980-06-12 1982-02-11 Asta-Werke Ag, Chemische Fabrik, 4800 Bielefeld "METHOD FOR PRODUCING NEW MUTANTS OF HERPES SIMPLEX VIRUS TYPE 1 AND TYPE 2"
US4767704A (en) * 1983-10-07 1988-08-30 Columbia University In The City Of New York Protein-free culture medium
NZ210501A (en) * 1983-12-13 1991-08-27 Kirin Amgen Inc Erythropoietin produced by procaryotic or eucaryotic expression of an exogenous dna sequence
CH652378A5 (en) 1983-12-21 1985-11-15 Heinrich Nagel Ag Wislikofen DEVICE FOR EXTRACTION AND CRUSHING IN A MEDIUM houses on gaseous of recoverable MATERIAL.
FI86885C (en) 1984-04-20 1992-10-26 Genentech Inc Method for Preparation of Human Recombinant Factor VIII and Nucleic Acid Sequences and Vectors Used thereto
WO1986004920A1 (en) 1985-02-13 1986-08-28 Biotechnology Research Partners, Limited Human metallothionein-ii promoter in mammalian expression system
US4978616A (en) * 1985-02-28 1990-12-18 Verax Corporation Fluidized cell cultivation process
EP0318487B1 (en) 1986-08-04 1993-10-13 The University Of New South Wales Serum free tissue culture medium containing polymeric cell-protective agent
US4710282A (en) 1986-08-08 1987-12-01 Maryan Chak Device for siliverizing running water
WO1988010269A1 (en) 1987-06-18 1988-12-29 Monash University Growth factors
AU627427B2 (en) 1987-06-30 1992-08-27 Amgen, Inc. Production of kallikrein
JP2507882B2 (en) 1988-02-17 1996-06-19 工業技術院長 Method for producing cell line with good growth independent of external growth factor
JPH01211878A (en) 1988-02-19 1989-08-25 Fujitsu Ltd Rehabilitation of i/o pin
US6048728A (en) * 1988-09-23 2000-04-11 Chiron Corporation Cell culture medium for enhanced cell growth, culture longevity, and product expression
US5573937A (en) * 1989-12-07 1996-11-12 Snow Brand Milk Products Co., Ltd. Serum free culture medium
SE465222C5 (en) 1989-12-15 1998-02-10 Pharmacia & Upjohn Ab A recombinant human factor VIII derivative and process for its preparation
DE69123140T2 (en) 1990-01-22 1997-04-03 The United States of America, represented by the Secretary, U.S. Department of Commerce, Washington, D.C. CO-INDEPENDENT NUTRITIONAL MEDIUM FOR THE HOLDING AND REPRODUCTION OF CELLS
JP2844484B2 (en) 1990-02-22 1999-01-06 味の素株式会社 Method for producing recombinant protein
JP2859679B2 (en) 1990-03-01 1999-02-17 協和醗酵工業株式会社 New cell line
US5378612A (en) * 1990-05-11 1995-01-03 Juridical Foundation The Chemo-Sero-Therapeutic Research Institute Culture medium for production of recombinant protein
JP2696001B2 (en) 1991-04-15 1998-01-14 財団法人化学及血清療法研究所 Medium for recombinant protein production
US5122469A (en) * 1990-10-03 1992-06-16 Genentech, Inc. Method for culturing Chinese hamster ovary cells to improve production of recombinant proteins
GB9022545D0 (en) 1990-10-17 1990-11-28 Wellcome Found Culture medium
JPH04228066A (en) 1990-10-23 1992-08-18 Rikagaku Kenkyusho Culture cell for expressing exogenote
US5213795A (en) * 1990-10-24 1993-05-25 Oxford Encephalomyocarditis virus vaccine
EP0531562A1 (en) * 1991-09-11 1993-03-17 Doerr, Hans-Wilhelm, Prof. Dr. med. Culturing of mammalian cells
JPH05123178A (en) 1991-11-01 1993-05-21 Ajinomoto Co Inc Production of l-phenylalanine
JP3511399B2 (en) * 1993-03-31 2004-03-29 株式会社三菱化学ヤトロン Cell culture substrate and cell culture method
DE4313620A1 (en) 1993-04-26 1994-10-27 Biotechnolog Forschung Gmbh Hamster cell lines and methods for glycoprotein recovery
AU7895898A (en) 1993-04-26 1998-10-08 Hans Wolf Mammal cell lines and method of obtaining glycoproteins
US5405637A (en) 1993-06-30 1995-04-11 Bristol-Myers Squibb Company Milk protein partial hydrolysate and infant formula containing same
JP4098372B2 (en) 1993-07-28 2008-06-11 三菱化学株式会社 Method for producing heparin-binding growth factor
JP2766165B2 (en) 1993-08-02 1998-06-18 株式会社バイオポリマー・リサーチ Method for producing bacterial cellulose
US5833162A (en) * 1993-12-22 1998-11-10 Daewoo Electronics Co., Ltd. Reel table driving device for a video cassette recorder
US5719050A (en) * 1993-12-24 1998-02-17 Eiken Chemical Co., Ltd. Animal cell culturing media containing N-acetyl-L-glutamic acid
EP0666312A1 (en) * 1994-02-08 1995-08-09 Wolfgang A. Renner Process for the improvement of mammalian cell growth
US5789247A (en) * 1994-04-01 1998-08-04 Ballay; Annick Expression in non-tumoral human lymphoblastoid lines with an integrative vector
EP0733100A1 (en) 1994-09-09 1996-09-25 Wolfgang A. Renner Chemical process for promoting the proliferation of animal cells
EP1213030B1 (en) * 1994-11-10 2009-04-22 Baxter Healthcare SA Method for producing biologicals in protein-free culture
US5753489A (en) * 1994-11-10 1998-05-19 Immuno Ag Method for producing viruses and vaccines in serum-free culture
AT403167B (en) 1994-11-14 1997-11-25 Immuno Ag SELECTION AND EXPRESSION OF FOREIGN PROTEINS BY MEANS OF A SELECTION-AMPLIFICATION SYSTEM
JP3244391B2 (en) 1994-12-08 2002-01-07 財団法人国際超電導産業技術研究センター Composite substrate and method for manufacturing single crystal substrate using the same
JPH10511082A (en) 1994-12-16 1998-10-27 ノバルティス・アクチエンゲゼルシャフト Production of recombinant secretory components
US5741705A (en) * 1995-02-23 1998-04-21 Quest International Flavors & Food Ingredients Company, Division Of Indopco, Inc. Method for in vitro cell growth of eucaryotic cells using low molecular weight peptides
EP0811056B1 (en) 1995-02-23 2000-05-31 Quest International B.V. Peptides for tissue and cell culture media
US5942386A (en) 1995-06-07 1999-08-24 Thomas Jefferson University Anti-fungal agents and methods of identifying and using the same
WO1996040866A1 (en) 1995-06-07 1996-12-19 Novartis Ag Serum-free media for primitive hematopoietic cells and methods of use thereof
AUPN442295A0 (en) 1995-07-26 1995-08-17 Commonwealth Scientific And Industrial Research Organisation Regulated autocrine growth of mammalian cells
US5851800A (en) * 1996-05-14 1998-12-22 Pharmacia & Upjohn Ab Process for producing a protein
AU4330597A (en) 1996-08-30 1998-03-19 Life Technologies, Inc. Serum-free mammalian cell culture medium, and uses thereof
US5804420A (en) 1997-04-18 1998-09-08 Bayer Corporation Preparation of recombinant Factor VIII in a protein free medium
EP1849860B1 (en) 1997-05-28 2010-11-17 Novartis Vaccines and Diagnostics S.r.l. Process for preparing an immunogenic factor of Corynebacterium diphtheriae using a culture medium with yeast extract as aminoacid source and no protein complexes of animal origin
AT407255B (en) * 1997-06-20 2001-02-26 Immuno Ag RECOMBINANT CELL CLONE WITH INCREASED STABILITY IN SERUM- AND PROTEIN-FREE MEDIUM AND METHOD FOR OBTAINING THE STABLE CELL CLONE
US6475725B1 (en) * 1997-06-20 2002-11-05 Baxter Aktiengesellschaft Recombinant cell clones having increased stability and methods of making and using the same
EP0986644B1 (en) 1997-07-23 2006-10-04 Boehringer Mannheim GmbH Production of erythropoietin by endogenous gene activation with viral promoters
JPH11211488A (en) 1998-01-21 1999-08-06 Matsushita Electric Ind Co Ltd Data transfer system using portable information terminal
WO1999057246A1 (en) 1998-05-01 1999-11-11 Life Technologies, Inc. Animal cell culture media comprising non-animal or plant-derived nutrients
AT409379B (en) 1999-06-02 2002-07-25 Baxter Ag MEDIUM FOR PROTEIN- AND SERUM-FREE CELL CULTURE
DK1200561T3 (en) 1999-08-05 2006-10-16 Baxter Ag Recombinant stable cell clone, its preparation and use
US6413744B1 (en) 1999-08-25 2002-07-02 Immunex Corporation Methods and host cells for improved cell culture
US7425437B2 (en) * 1999-11-26 2008-09-16 Crucell Holland B.V. Vaccines against West Nile Virus
JP5063852B2 (en) 2000-09-25 2012-10-31 ポリマン サイエンティフィック イミューンバイオロジッシュ フォーシュング ゲゼルシャフト ミット ベシュレンクテル ファフツング Live vaccine and production method
EP1208966A1 (en) 2000-11-27 2002-05-29 Cheng-Kun Liao Manufacturing process of patio tabletop glass with broken protection
ES2398706T3 (en) 2002-07-09 2013-03-21 Baxter International Inc. Animal protein free medium for cell culture
JP4316484B2 (en) 2004-12-10 2009-08-19 シャープ株式会社 Image forming apparatus, toner density control method, toner density control program and recording medium therefor

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998015614A1 (en) * 1996-10-10 1998-04-16 Life Technologies, Inc. Animal cell culture media comprising plant-derived nutrients
WO1999047648A2 (en) * 1998-03-13 1999-09-23 Aventis Pasteur Medium and method for viral propagation and growth
WO2000003000A2 (en) * 1998-07-10 2000-01-20 Chugai Seiyaku Kabushiki Kaisha Serum-free medium for culturing animal cells
JP2001211878A (en) * 2000-02-04 2001-08-07 National Federation Of Dairy Cooperative Associations Method of culturing yogurt starter lactobacillus in high concentration, and method of producing yogurt from the obtained high concentration culture solution

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BUTLER; SPIER; GRIFFITHS, ANIMAL CELL BIOTECHNOLOGY, vol. 3, 1988, pages 283 - 303
PATENT ABSTRACTS OF JAPAN vol. 2000, no. 25 12 April 2001 (2001-04-12) *

Cited By (59)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8084252B2 (en) 1997-06-20 2011-12-27 Baxter Innovations Gmbh Recombinant cell clones having increased stability and methods of making and using the same
USRE46897E1 (en) 1997-06-20 2018-06-19 Baxalta Incorporated Recombinant cell clones having increased stability and methods of making and using the same
USRE46860E1 (en) 1997-06-20 2018-05-22 Baxalta Incorporated Recombinant cell clones having increased stability and methods of making and using the same
USRE46745E1 (en) 1997-06-20 2018-03-06 Baxalta Incorporated Recombinant cell clones having increased stability and methods of making and using the same
US8080414B2 (en) 1997-06-20 2011-12-20 Baxter Innovations Gmbh Recombinant cell clones having increased stability and methods of making and using the same
US8084251B2 (en) 1997-06-20 2011-12-27 Baxter Innovations Gmbh Recombinant cell clones having increased stability and methods of making and using the same
US8329465B2 (en) 1997-06-20 2012-12-11 Baxter Innovations Gmbh Recombinant cell clones having increased stability and methods of making and using the same
US8021881B2 (en) 1999-09-28 2011-09-20 Baxter Innovations Gmbh Medium for the protein-free and serum-free cultivation of cells
US9441203B2 (en) 1999-09-28 2016-09-13 Baxalta Innovations Gmbh Medium for the protein-free and serum-free cultivation of cells
US8722406B2 (en) 1999-09-28 2014-05-13 Baxter Innovations Gmbh Medium for the protein-free and serum-free cultivation of cells
US9163211B2 (en) 2002-07-09 2015-10-20 Baxter International Inc. Animal protein free media for cultivation of cells
US8524497B2 (en) 2002-07-09 2013-09-03 Baxter International Inc. Animal protein free media for cultivation of cells
US7955833B2 (en) 2002-07-09 2011-06-07 Baxter International Inc. Animal protein free media for cultivation of cells
WO2005083058A1 (en) * 2004-03-01 2005-09-09 Ares Trading S.A. Use of a serum-free cell culture medium for the production of il-18bp in mammalian cells
US7553665B2 (en) 2004-03-01 2009-06-30 Ares Trading S.A. Process for the cultivation of mammalian cells producing IL-18BP in serum-free cell culture medium
US8221969B2 (en) 2004-04-19 2012-07-17 Sanyo Chemical Industries, Ltd. Method of producing virus
EP1739167A4 (en) * 2004-04-19 2008-05-07 Denka Seiken Kk Method of producing virus
EP1739167A1 (en) * 2004-04-19 2007-01-03 DENKA SEIKEN Co., Ltd. Method of producing virus
JP2008512443A (en) * 2004-09-09 2008-04-24 カイロン ベーリング ゲーエムベーハー アンド カンパニー Reduce potential iatrogenic risk associated with influenza vaccines
JP2011207911A (en) * 2004-09-09 2011-10-20 Novartis Vaccines & Diagnostics Gmbh & Co Kg Decreasing potential iatrogenic risk associated with influenza vaccine
EP2213726A1 (en) * 2004-10-29 2010-08-04 Baxter International Inc. Animal protein-free media for cultivation of cells
US8440408B2 (en) 2004-10-29 2013-05-14 Baxter International Inc. Animal protein-free media for cultivation of cells
US9222075B2 (en) 2004-10-29 2015-12-29 Baxalta Incorporated Animal protein-free media for cultivation of cells
EP2218776A1 (en) * 2004-10-29 2010-08-18 Baxter International Inc. Animal protein-free media for cultivation of cells
EP2213725A1 (en) * 2004-10-29 2010-08-04 Baxter International Inc. Animal protein-free media for cultivation of cells
EP2213724A1 (en) * 2004-10-29 2010-08-04 Baxter International Inc. Animal protein-free media for cultivation of cells
JP4847962B2 (en) * 2004-10-29 2011-12-28 バクスター・インターナショナル・インコーポレイテッド Cell culture medium without animal protein
US9809796B2 (en) 2004-10-29 2017-11-07 Baxalta GmbH Animal protein-free media for cultivation of cells
CN101065480B (en) * 2004-10-29 2010-05-26 巴克斯特国际公司 Animal protein-free media for cultivation of cells
WO2006045438A1 (en) * 2004-10-29 2006-05-04 Baxter International Inc. Animal protein-free media for cultivation of cells
US10138461B2 (en) 2004-10-29 2018-11-27 Baxalta GmbH Animal protein-free media for cultivation of cells
AU2012238257B2 (en) * 2004-10-29 2014-04-10 Takeda Pharmaceutical Company Limited Animal Protein-Free Media for Cultivation of Cells
US10655099B2 (en) 2004-10-29 2020-05-19 Baxalta Incorporated Animal protein-free media for cultivation of cells
US8748156B2 (en) 2004-10-29 2014-06-10 Baxter International Inc. Animal protein-free media for cultivation of cells
US9714411B2 (en) 2004-10-29 2017-07-25 Baxalta GmbH Animal protein-free media for cultivation of cells
KR100679112B1 (en) 2004-11-17 2007-02-07 삼성정밀화학 주식회사 Method for Animal Cell Culture
JP4693839B2 (en) * 2005-03-10 2011-06-01 共立製薬株式会社 Cell line that can be cultured without animal-derived components, method for producing the same, virus production method using the same, and vaccine production method
JPWO2006095431A1 (en) * 2005-03-10 2008-08-14 共立製薬株式会社 Cell line that can be cultured without animal-derived components, method for producing the same, virus production method using the same, and vaccine production method
US7910366B2 (en) 2005-03-10 2011-03-22 Kyoritsu Seiyaku Corporation Cell strain capable of being cultured without ingredients derived from animals, method of producing the same, method of producing virus using the same, and method of producing vaccine
JP2008536499A (en) * 2005-04-13 2008-09-11 メリアル リミテッド Porcine circovirus production assay
JP2007000077A (en) * 2005-06-23 2007-01-11 Hitachi Medical Corp Method for serum-free culture of adherent animal cell and culture medium for serum-free culture of adherent animal cell
EP1974014B1 (en) 2006-01-04 2017-04-19 Baxalta Incorporated Oligopeptide-free cell culture media
EP3121266B1 (en) 2006-01-04 2020-02-26 Baxalta Incorporated Oligopeptide-free cell culture media
US9758568B2 (en) 2006-01-04 2017-09-12 Baxalta GmbH Oligopeptide-free cell culture media
US10696731B2 (en) 2006-01-04 2020-06-30 Baxalta GmbH Oligopeptide-free cell culture media
EP2017333A4 (en) * 2006-04-28 2009-04-29 Kyoritsu Seiyaku Corp Feline cell capable of being cultured without animal protein, and method for production of virus and method for production of vaccine using the feline cell
US7767451B2 (en) 2006-04-28 2010-08-03 Kyoritsu Seiyaku Corporation Feline cell capable of being cultured without animal-derived protein, and method for producing virus and method for producing vaccine using thereof
EP2017333A1 (en) * 2006-04-28 2009-01-21 Kyoritsu Seiyaku Corporation Feline cell capable of being cultured without animal protein, and method for production of virus and method for production of vaccine using the feline cell
WO2015086598A3 (en) * 2013-12-09 2015-10-01 Vibalogics Gmbh Method of culturing vero cells
WO2015101510A1 (en) * 2013-12-30 2015-07-09 Baxter Healthcare Sa A method of predicting a performance characteristic of a plant or yeast hydrolysate and its use
US11650159B2 (en) 2013-12-30 2023-05-16 Takeda Pharmaceutical Company Limited Method of predicting a performance characteristic of a plant or yeast hydrolysate and its use
US10527551B2 (en) 2013-12-30 2020-01-07 Baxalta Incorporated Method of predicting a performance characteristic of a plant or yeast hydrolysate and its use
EP3152317B1 (en) 2014-06-04 2019-01-02 Amgen Inc. Methods for harvesting mammalian cell cultures
US11384378B2 (en) 2014-06-04 2022-07-12 Amgen Inc. Methods for harvesting mammalian cell cultures
US11427848B2 (en) 2014-06-04 2022-08-30 Amgen Inc. Methods for harvesting mammalian cell cultures
CN105385661B (en) * 2014-09-03 2019-02-05 普莱柯生物工程股份有限公司 A kind of method and its application of porcine circovirus 2 type large-scale culture
CN105385661A (en) * 2014-09-03 2016-03-09 普莱柯生物工程股份有限公司 Porcine circovirus type 2 large-scale cultivation method and applications thereof
CN105154389A (en) * 2015-10-15 2015-12-16 南京三生生物技术有限公司 Low-serum protein-free culture medium suitable for PK-15 cell growth and preparation method of culture medium
EP4093848A4 (en) * 2020-01-24 2024-08-07 Air Protein Inc Microorganism-derived protein hydrolysates, and methods of preparation and use thereof

Also Published As

Publication number Publication date
PT2287288E (en) 2012-12-10
EP1520008A1 (en) 2005-04-06
CN1681917A (en) 2005-10-12
ES2398706T3 (en) 2013-03-21
US20040077086A1 (en) 2004-04-22
ES2394085T3 (en) 2013-01-17
US9163211B2 (en) 2015-10-20
PL214284B1 (en) 2013-07-31
CN101058800B (en) 2013-03-13
JP5485863B2 (en) 2014-05-07
JP2005532057A (en) 2005-10-27
JP2012034712A (en) 2012-02-23
JP2011041580A (en) 2011-03-03
US7955833B2 (en) 2011-06-07
HK1108713A1 (en) 2008-05-16
JP2015083005A (en) 2015-04-30
EP2287288B1 (en) 2012-11-07
EP2287288A1 (en) 2011-02-23
CN100591759C (en) 2010-02-24
PT1520008E (en) 2012-09-26
NZ538094A (en) 2007-01-26
HK1083635A1 (en) 2006-07-07
US20110217772A1 (en) 2011-09-08
JP6244294B2 (en) 2017-12-06
AU2003249990A1 (en) 2004-01-23
SI1520008T1 (en) 2012-12-31
JP6178109B2 (en) 2017-08-09
JP2013172741A (en) 2013-09-05
DK2287288T3 (en) 2013-01-07
MXPA05000418A (en) 2005-03-23
EP1520008B1 (en) 2012-09-05
AU2003249990B2 (en) 2007-06-28
PL374782A1 (en) 2005-10-31
DK1520008T3 (en) 2012-10-15
US8524497B2 (en) 2013-09-03
CA2491992A1 (en) 2004-01-15
US20130316434A1 (en) 2013-11-28
JP6084359B2 (en) 2017-02-22
CN101058800A (en) 2007-10-24

Similar Documents

Publication Publication Date Title
US9163211B2 (en) Animal protein free media for cultivation of cells
EP2213725B1 (en) Animal protein-free media for cultivation of cells
DK1974014T3 (en) OLIGOPEPTID-FREE CELL CULTURE MEDIA

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWE Wipo information: entry into national phase

Ref document number: 2491992

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 42/DELNP/2005

Country of ref document: IN

WWE Wipo information: entry into national phase

Ref document number: PA/a/2005/000418

Country of ref document: MX

Ref document number: 2004518745

Country of ref document: JP

WWE Wipo information: entry into national phase

Ref document number: 374782

Country of ref document: PL

WWE Wipo information: entry into national phase

Ref document number: 2003762657

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 2003249990

Country of ref document: AU

WWE Wipo information: entry into national phase

Ref document number: 538094

Country of ref document: NZ

WWE Wipo information: entry into national phase

Ref document number: 20038213265

Country of ref document: CN

WWP Wipo information: published in national office

Ref document number: 2003762657

Country of ref document: EP

WWG Wipo information: grant in national office

Ref document number: 2003249990

Country of ref document: AU