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WO2008035631A1 - Procédé de production d'une substance - Google Patents

Procédé de production d'une substance Download PDF

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Publication number
WO2008035631A1
WO2008035631A1 PCT/JP2007/067941 JP2007067941W WO2008035631A1 WO 2008035631 A1 WO2008035631 A1 WO 2008035631A1 JP 2007067941 W JP2007067941 W JP 2007067941W WO 2008035631 A1 WO2008035631 A1 WO 2008035631A1
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WIPO (PCT)
Prior art keywords
dipeptide
culture
cell
cells
medium
Prior art date
Application number
PCT/JP2007/067941
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English (en)
Japanese (ja)
Inventor
Yoshinobu Konno
Kentaro Sakai
Masakazu Takagishi
Ken Takahashi
Shinji Sakae
Masamichi Koike
Toshiyuki Suzawa
Shinji Hosoi
Yasufumi Imamoto
Hisaya Tanaka
Original Assignee
Kyowa Hakko Kogyo Co., Ltd.
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Application filed by Kyowa Hakko Kogyo Co., Ltd. filed Critical Kyowa Hakko Kogyo Co., Ltd.
Publication of WO2008035631A1 publication Critical patent/WO2008035631A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0018—Culture media for cell or tissue culture
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/998—Proteins not provided for elsewhere

Definitions

  • the present invention relates to a method for culturing animal cells having an ability to produce a substance, characterized by adding a peptide and culturing in an animal cell culturing method having an ability to produce a substance,
  • a method for producing a substance by culturing animal cells having the ability to produce a substance the dipeptide is added and cultured, the substance is produced and accumulated in the culture, and the substance is removed from the culture.
  • a method for producing a substance characterized by collecting, and culturing by adding a dipeptide to an animal cell having the ability to produce the substance, and determining the productivity per cell of the substance produced from the cell.
  • a method for maintaining the viability of a cell a method for maintaining the viability of the substance, characterized by adding a dipeptide to an animal cell having an ability to produce a substance and culturing the animal cell.
  • dipeptide A method for suppressing an increase in the concentration of ammonium ions in the culture medium of the cell, and a method of culturing by adding a dipeptide to an animal cell having the ability to produce a substance. And a method for inhibiting apoptosis of the cell.
  • Non-patent Document 1 Among glycoproteins, antibodies have recently been approved as various pharmaceuticals, and many antibodies are under development (Non-patent Document 1). In the future, it is predicted that production of useful substances such as antibodies in animal cells is expected to become active S, and that the productivity of useful substances in animal cells is sufficiently high! / RU
  • CHO cells Chinese hamster ovary tissue-derived cells
  • hypridoma as host cells that produce antibodies and other glycoprotein substances is produced from such cells. This is because the sugar chains bound to the glycoproteins are relatively close to humans! (Patent Documents 1 and 2).
  • Cells use glucose and glutamine as energy sources.
  • glucose or glutamine is added to a medium used for cell culture and the cells are cultured, ammonia generated by the metabolism of glutamine by the cells accumulates in the medium.
  • Glutamine in the medium Ammonia generated by natural decomposition of the water also accumulates in the medium. Ammonia is known to affect the growth of animal cells and substances produced by animal cells (Non-Patent Documents 2 and 3).
  • Non-Patent Documents 2 and 3 As a method for reducing the accumulation of ammonia in the medium due to cell metabolism, it is known to construct cells that do not require glutamine as a substrate (Patent Document 3).
  • a basal medium supplemented with L-amino acid L-glutamine is known for the purpose of preventing the decomposition of glutamine due to heat sterilization of the medium (Patent Document 4).
  • L-alanil-L-glutamine is known to have higher thermal stability than L-glutamine, and is used in infusion preparations (Non-patent Documents 4 and 5).
  • Culture method of Hela cells using basal medium supplemented with L-alanil-L-glutamine (Patent Document 5), basal medium containing 300 mg / L L-alanil-L-glutamine for human cell culture Known (Patent Document 6).
  • a medium containing L-alanil-L-glutamine is also known as a cell freezing medium (Non-patent Document 6).
  • L-alanil-L-glutamine is sold as a supplement from Invitrogen, LLC H Biosciences, and it is known to suppress the generation of ammonia by adding the supplement to the basic medium (non- Patent Document 7).
  • Non-patent Document 8 it is known that conditioned cells are required because not all cells grow in a medium containing L-alanil-L-daltamin!.
  • Non-patent Document 9 As a dipeptide containing L-tyrosine, L-barilloux L-tyrosine, which is considered to have a blood pressure effect, is known (Non-patent Document 9).
  • Patent Document 1 W096 / 39488
  • Patent Document 2 US4724206
  • Patent Document 3 WO87 / 04462
  • Patent Document 4 JP-A 61-271985
  • Patent Document 5 EP0220379
  • Patent Document 6 US 5328844
  • Non-Patent Document 1 Nature Rev. Drug. Discov., 3, 383 (2004)
  • Non-Patent Document 2 Gastroenterology, 107, 429 (1994)
  • Non-Patent Document 3 Biotechnology Prog, 18, 3129 (2002)
  • Non-Patent Document 4 Biotechnology Bioeng, 68, 370 (2000)
  • Non-Patent Document 5 Infusionsther Transfusioned, 22, 317 (1995)
  • Non-Patent Document 6 Biotechnology Prog., 20, 1113 (2004)
  • Non-Patent Document 7 Quest, 1, 7 (2004)
  • Non-Patent Document 8 Biotechnology Bioeng, 64, 298 (1999)
  • Non-Patent Document 9 Hypertension Research, 28, 545 (2005)
  • the present invention provides the following (1) to (90).
  • a method for cultivating animal cells having the ability to produce a substance comprising culturing with the addition of a dipeptide.
  • a method for producing a substance by culturing animal cells having the ability to produce a substance the dipeptide is added and cultured, the substance is produced and accumulated in the culture, and the culture is used to produce the substance.
  • a method for producing a substance comprising collecting the substance.
  • a method for improving productivity per cell of a substance produced from the cell which comprises culturing by adding a dipeptide to an animal cell having the ability to produce the substance.
  • a method for maintaining the survival rate of a cell which comprises culturing an animal cell having the ability to produce a substance by adding a dipeptide.
  • (61) A method of suppressing an increase in the concentration of ammonium ions in a culture solution of a cell, characterized by adding a dipeptide to a cell having an ability to produce a substance and culturing the animal cell. How to make it.
  • a method for suppressing early apoptosis of a cell which comprises culturing an animal cell having the ability to produce a substance by adding a dipeptide.
  • the present invention relates to a method for culturing animal cells having the ability to produce a substance characterized by adding a dipeptide and culturing the animal cell in a method for culturing animal cells having the ability to produce a substance.
  • a method for producing a substance by culturing animal cells having the ability to cultivate, culturing with addition of a dipeptide, producing and accumulating the substance in the culture, and collecting the substance from the culture A method for improving the per-cell productivity of a substance produced from the cell, characterized by adding a dipeptide to an animal cell having the ability to produce the substance and culturing the animal cell Maintaining the viability of the cells, characterized by adding a dipeptide to an animal cell capable of producing a substance and culturing the cells.
  • a method for suppressing an increase in the concentration of ammonium ions in a culture solution of the cell and a method for producing the substance, characterized by adding a dipeptide to an animal cell capable of producing the substance and culturing the animal cell
  • a method for suppressing apoptosis of an animal cell which comprises culturing by adding a dipeptide to an animal cell having the ability.
  • FIG.l CHO cells
  • Figure 1 shows the time course of viable cell density (cells / mL) when batch culture was performed in Erlenmeyer flasks using BP-8472).
  • the mouth in the figure indicates Gin culture, and the country indicates AlaGln culture.
  • FIG. 2 shows changes over time in ammonium ion concentration (mmol / L), viability (%), and viable cell density (cells / mL) when fed batch culture with 2L bioreactor using BP-8472). Shown in The mouth in the figure indicates Gin Gin culture, and the country indicates Gin-AlaGln culture.
  • FIG. 3 shows the time-dependent change in the density of living cells (cells / mU over time) when FE cell culture was performed in an Erlenmeyer flask using the CHO cell line Ms704 / CD20 (FERM BP-10092). Mouth indicates Gin-Gin culture, ⁇ indicates Gin-AlaGln culture, and country indicates A1 aGln-AlaGln culture.
  • Fig. 4 shows the protein concentration (mg / mL, SPR g / 10 6 cells / day) when FE cells were cultured in Erlenmeyer flasks using CHO cells Ms704 / CD20 (FERM BP-10092). Shown in 4. The mouth in the figure indicates Gin-Gin culture, the hatched line indicates Gin-AlaGln culture, and the country indicates AlaGln-AlaGln culture.
  • FIG 5 CHO cells Ms704 / CD20 strain when (FERM BP- 10092) was performed Fuedobatchi culture in 2L Baioria Kuta one using, viable cell density (cells / m L), Anmoniu Ion Concentration (mmol / L Figure 5 shows the changes over time. The mouth in the figure indicates Gin-Gin culture, ⁇ indicates Gin-AlaGln culture, and the country indicates AlaGln-AlaGln culture.
  • FIG. 6 Protein concentration (mg / L), SPR g / 10 6 cells / day when FE cells were cultured in 2L bioreactor using CHO cell line Ms704 / CD20 (FERM BP-10092) The percentage of early apoptotic cells (%) is shown in FIG. The mouth in the figure indicates Gin-Gin culture, the hatched line indicates Gin-AlaGln culture, and the country indicates AlaGln-AlaGln culture. The
  • FIG. 7 L glutamine concentration (mmol / L) in culture medium when fed batch culture with 2L bioreactor using CHO cell line Ms704 / CD20 (FERM BP-10092), L-alanil-L-glutamine
  • ⁇ indicates Gin-AlaGln culture
  • country indicates AlaGln-AlaGln culture.
  • the dotted line in the figure indicates the L-glutamine concentration
  • the solid line indicates the L-alanyl-L-glutamine concentration.
  • FIG. 8 shows SPR g / 10 6 cells / day when fedbatch culture was performed in an Erlenmeyer flask using CHO cell strain Ms704 / CD20 (FERM BP-10092).
  • the mouth in the figure indicates Tyr culture, and the country indicates AlaTyr culture.
  • Fig. 9 shows the time course of viable cell density (cells / mU) when FE cell culture in an Erlenmeyer flask using the CHO cell line Ms704 / CD20 (FERM BP-10092) was performed.
  • ⁇ indicates culture without addition of ammonium chloride
  • mouth indicates culture with addition of ammonium chloride 1 Ommol / L
  • ⁇ indicates culture with addition of ammonium chloride 20 mmol / L
  • X indicates culture with addition of ammonium chloride 30 mmol / L * Indicates culture with addition of ammonium chloride 40 mmol / L
  • ⁇ indicates culture with addition of ammonium chloride 50 mmol / L
  • + indicates culture with addition of ammonium chloride 60 mmol / L.
  • FIG. 10 shows the specific growth rate QT 1 ) on the 4th day of culture when food batch culture was performed in triangular flasks using the CHO cell line Ms704 / CD20 (FERM BP-10092).
  • the country in the figure shows the culture without ammonium chloride, and the mouth shows the culture with ammonium chloride 10 mol / L, 20 mol / L, 30 mol / L, 40 mol / L, 50 mol / L, and 60 mol / L. .
  • FIG. Ll Changes in viable cell density (cells / mU, protein concentration (mg / L) over time when FE cells were cultured in 2L bioreactor using CHO cell line Ms704 / CD20 (FERM BP-10092).
  • Figure 11 shows the culture without addition of ammonium chloride
  • ⁇ shows the culture with addition of ammonium chloride 24 mmol / L.
  • the present invention relates to a method for culturing animal cells having the ability to produce a substance.
  • the present invention relates to a method for culturing animal cells, characterized by adding a cell and culturing.
  • the dipeptide used in the present invention is not particularly limited, and examples include dipeptides containing L-glutamine, dipeptides containing L-tyrosine, and the like. Dipeptides containing L-glutamine are preferably alanylglutamine. L-alanil L-glutamine is particularly preferred.
  • L-alanyl-L tyrosine which is preferably alanyl tyrosine, is particularly preferable.
  • a dipeptide containing L-glutamine can be produced by a known synthesis method, enzyme method or fermentation method.
  • a method for producing L-alanyl mono L-glutamine synthetic methods [Organic Process Research & Development 5, 132 (2001), JP 6-234715] and fermentation methods (WO2004 / 058960, WO2006 / 001379) S.
  • a dipeptide containing L-tyrosine can also be produced by a known synthesis method, enzyme method, or fermentation method.
  • methods for producing L-alanyl-L-tyrosine include synthesis methods and fermentation methods (WO2004 / 058960, WO2006 / 001379) and the like S.
  • the method of adding the dipeptide to the medium is not particularly limited! /, But it is preferable to add it to the basic medium and / or the feed medium to culture animal cells! / .
  • the dipeptide When added as a feed medium, the dipeptide may be added to the medium alone as the feed medium, or the dipeptide and other medium components may be added to the medium as a premixed feed medium. It may be added. Further, the feed medium may be divided and added to the medium discontinuously, or the feed medium may be continuously added to the medium.
  • the concentration of the dipeptide added to the medium may be appropriately selected depending on the type of animal cells used for culture, the type of substance to be produced, the type of dipeptide, the timing of addition of the dipeptide, etc., but preferably the final concentration is 0.01. To 1000 mmol / L, more preferably 0.1 to 500 mmol / L, and particularly preferably 1 to 100 mmol / L.
  • the final concentration of dipeptide refers to the concentration of the dipeptide in the medium immediately after being added to the medium.
  • the timing of adding the dipeptide is preferably added to the medium in the logarithmic growth phase, particularly preferably in the first half of the logarithmic growth phase.
  • the logarithmic growth phase varies depending on the cell line to be cultured and the culture control method. This refers to the period until the time when the number of cells to be obtained becomes constant.
  • the method for storing the feed medium is not particularly limited as long as the medium is maintained in a sterile state, and examples thereof include a method using a stainless steel tank and a disposable bag.
  • the medium used in the present invention may be any medium as long as it can be used for animal cell culture, and a basal medium used for normal animal cell culture is used. Glutamine and / or tyrosine may be previously contained as a medium component, or may not be contained.
  • the ability to use any medium such as a serum-containing medium, a medium that does not contain animal-derived substances such as serum albumin and serum fractions, a serum-free medium, a protein-free medium, etc. It is preferable to use a medium or a protein-free medium.
  • a basal medium used for normal animal cell culture used in the method of the present invention for example, RPMI1640 medium [The Journal of the American Medical
  • EX—CELL TM 302 medium manufactured by GIA LEUTI Biosciences or their modified or mixed medium, preferably RPMI1640 medium, DMEM medium, F12 medium, IMDM medium and EX—CEL
  • L TM 302 medium or the like is used.
  • serum-free medium a basal medium containing a physiologically active substance, a nutrient factor, a carbon source that can be assimilated by animal cells, a nitrogen source and the like instead of serum is used.
  • additives necessary for the growth of animal cells to the serum-free medium as needed. These additives are preferably contained in the medium in advance before culturing.
  • Examples of nutritional factors include glucose, amino acids, vitamins and the like.
  • Examples of amino acids include L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cystine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-sip Icin, L-lysine , L-methionine, L-phenenolealanine, L-porporin, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, etc., which can be used alone or in combination. Also, solvates such as these salts and / or hydrates may be used.
  • vitamins include D biotin, D pantothenic acid, choline, folic acid, myo inositol, niacinamide, pyridoxal, riboflavin, thiamine, cyanocobalamin, DL-a tocopherol, etc., one or a combination of two or more Used. Also, solvates such as these salts and / or hydrates may be used! /.
  • physiologically active substances include insulin, transferrin, serum albumin, serum fractions containing growth factors, and the like.
  • substances added in place of animal-derived substances include physiologically active substances produced by genetic recombination methods, hydrolysates or animal-derived substances. And lipids.
  • hydrolyzate examples include hydrolysates such as soybean, wheat, rice, peas, cottonseed or yeast extract.
  • lipids that do not contain animal-derived substances include cholesterol, linoleic acid, linolenic acid, and the like. Also included are solvates such as salts and / or hydrates thereof.
  • ADPF medium (Animal derived protein free medium; manufactured by High Clone), CD—Hybridoma medium (Invitrogen), CD—CHO medium (Invitrogen), IS—CD—CHO medium (IS) Etc.).
  • a medium containing a high concentration of amino acids and vitamins for example, a medium in which RPMI1640 medium, DMEM medium and F12 medium are mixed at a ratio of 1: 1: 1, A medium in which DMEM medium and F12 medium are mixed at a ratio of 1: 1, hybridoma SFM medium (manufactured by Invitrogen) and the like are preferably used.
  • animal cells having the ability to produce the substances used in the present invention include cells belonging to any of mammals, birds, reptiles, amphibians, fish and insects.
  • animal cells belonging to mammals are preferably used, more preferably animal cells derived from humans or primates such as monkeys or animal cells derived from rodents such as mice, rats or hamsters.
  • Examples of cells belonging to mammals include myeloma cells or cells derived from myeloma cells, ovarian cells, kidney cells, blood cells, uterine cell connective tissue cells, mammary cells or embryonic retinoblasts.
  • myeloma cells or cells derived from myeloma cells and ovary cells are preferred.
  • the substance to be produced is an antibody, it is preferably an antibody-producing cell such as a hyperidoma.
  • Examples of cells belonging to mammals include human cell lines HL-60 (ATCC CCL-240), HT-1080 (ATCC CCL-121), HeLa (ATCC CCL-2), 293 (E CACC 85120602 ), Namalwa (ATCC CRL—1432), Namalwa KJM—1 (Cytotechnology, 1, 151 (1988), NM—F9 cells (DSM ACC2605, WO05 / 17130) and PER. C6 cells (ECACC No.
  • the cell lines VERO ATCC CCL-1651) and COS-7 (ATCC CRL-1651), the mouse cell lines C127I (ATCC CRL-1616), Sp2 / 0— Agl4 (ATC C CRL— 1581) NIH3T3 (ATCC CRL— 1658), NS0 (ATCC CRL—182 7), rat cell lines Y3 Agl.
  • VERO ATCC CCL-1651
  • COS-7 ATCC CRL-1651
  • the mouse cell lines C127I ATCC CRL-1616
  • Sp2 / 0— Agl4 ATC C CRL— 1581
  • NIH3T3 ATCC CRL— 1658
  • NS0 ATCC CRL—182 7
  • rat cell lines Y3 Agl Y3 Agl.
  • Examples include Spodoptera frugiperda cell line Sf9 (ATCC CRL-1711), etc.
  • primary monkey kidney cells, primary rabbit kidney cells, primary chicken embryos are used as primary culture cells for vaccine production. Cells, primary quail fetal cells, and the like.
  • Myeloma cells or cells derived from myeloma cells include Sp2 / 0—Agl4, NS0, Y3 Agl. 2. 3., YO or YB2 / 0.
  • Examples of ovarian cells or cells derived from ovarian cells include CHO—Kl, CHO / dhfr_, or CHO / DG44 isostatic S.
  • 293, VERO, COS-7, BHK21 or M DCK etc. are used for kidney cells
  • HL-60, Namalwa, Namalwa KJM-1 or NM-F9 etc. are used for blood cells
  • HeLa isotonic is used for uterine cells.
  • the connective tissue cell is HT-1080 or NIH3T3 isobaric mammary cell is C1271I isobaric embryonic retinoblast is PER. C6 and so on.
  • Animal cells having the ability to produce a substance used in the present invention include animal cells transformed with a recombinant vector containing a gene involved in the production of the substance, and a substance that is subjected to mutation treatment to produce the substance.
  • Hydridoma which is a fused cell of an antibody-producing cell such as a B cell or an antibody-producing cell and a myeloma cell. Animal cells and the like that have been subjected to a mutation treatment that increases the expression level of the substance are also included in the animal cells of the present invention.
  • Examples of cells that have been subjected to mutation treatment to produce substances include cells in which mutations have been introduced into protein modifying enzymes to enable production of desired substances.
  • the desired substance is a glycoprotein
  • cells in which mutations are introduced into various sugar chain-modifying enzymes may be used in order to change the structure of the sugar chain that binds to the protein. .
  • a cell transformed with a recombinant vector containing a gene involved in the production of the substance introduces the recombinant vector containing the DNA involved in the production of the substance and a promoter into the animal cell used in the present invention. Can be obtained.
  • DNA involved in the production of a substance for example, DNA encoding a substance such as a peptide, DNA encoding an enzyme or protein involved in the biosynthesis of the substance, and the like can be used.
  • the substance produced by the method of the present invention may be any substance that can produce animal cells, but is preferably a substance that can produce animal cells belonging to mammals, and examples thereof include peptides.
  • substances produced by the method of the present invention include biocatalytic molecules such as ribozyme, keratin, collagen, elastin, resilin, and hive mouth-in structures.
  • Vaccine such as formation / retention molecule, pressure ulcer vaccine, polio vaccine, measles vaccine, rubella vaccine, mumps vaccine, rabies vaccine, varicella vaccine, ushi epidemic fever vaccine, Ibaraki disease cutin and ushi infectious tracheitis vaccine Or viruses such as adenovirus and baculovirus.
  • Peptides produced by the method of the present invention include peptides derived from eukaryotic cells, preferably peptides derived from mammalian cells.
  • a peptide having physiological activity is preferred.
  • the peptide may be in any form as long as the desired peptide is contained.
  • the peptide may be an artificially modified peptide such as a fusion peptide fused with another peptide. It may be a partial fragment.
  • the peptides produced by the method of the present invention include glycoproteins.
  • glycoproteins include antibodies, erythropoietin (EPO). Biol. Chem., 252. 5558 (1977)], thrombopoietin ( ⁇ ) [Nature, 369. 533].
  • EPO erythropoietin
  • ⁇ thrombopoietin
  • Tissue type plasminogen activator t-PA
  • plow mouth kinase plow mouth kinase
  • thrombomodulin antithrombin
  • protein blood coagulation factor VII blood coagulation factor VIII
  • blood coagulation factor IX blood coagulation factor X
  • Blood coagulation factor XI blood coagulation factor XII
  • prothrombin complex fibrinogen, albumin, gonadotropin, thyroid stimulating hormone, epidermal growth factor (EGF), hepatocyte growth factor (HGF), keratinocyte growth factor, activin, bone Forming factor, granulocyte colony stimulating factor (G—CSF) U. Biol. Chem., 25 8, 9017 (1983)], macrophage colony stimulating factor (M—CSF).
  • EGF epidermal growth factor
  • HGF hepatocyte growth factor
  • G—CSF granulocyte colony stimulating factor
  • the antibody may be any antigen-binding antibody, such as an antibody that binds to a tumor-related antigen, an antibody that binds to an antigen related to allergy or inflammation, an antibody that binds to an antigen related to cardiovascular disease, It is preferably an antibody that binds to an antigen associated with an autoimmune disease, or an antibody that binds to an antigen associated with a virus or bacterial infection, and the antibody class is preferably IgG.
  • the antibody produced by the method of the present invention includes a fragment containing a part of the antibody, such as Fab (abbreviation of Fragment of antigen binding), Fab, F (ab,),
  • Examples include single chain antibodies (single chain Fv; hereinafter referred to as scFv), disulfide stabilized antibodies (hereinafter referred to as dsFv), and fusion proteins containing the Fc region of antibodies.
  • scFv single chain Fv
  • dsFv disulfide stabilized antibodies
  • antibodies include antibodies produced by gene recombination techniques, in other words, antibody genes, in addition to antibodies secreted by hyperoma cells produced from spleen cells of immunized animals after immunization of animals with antigens.
  • an antibody obtained by introducing an antibody expression vector into which a vector has been introduced into a host cell.
  • Specific examples include antibodies produced by Hypridoma, human-type chimerized antibodies, humanized antibodies or human antibodies.
  • the human chimeric antibody is a non-human animal antibody heavy chain variable region (hereinafter, the heavy chain is also referred to as H chain, the variable region is also referred to as HV or VH) and an antibody light chain variable region (hereinafter referred to as "H chain”).
  • the light chain is also referred to as LV or VL as the light chain) and the heavy chain constant region of human antibodies (hereinafter, the constant region is also referred to as CH! /)
  • the light chain constant region of human antibodies hereinafter also referred to as CL! /, U
  • CL! /, U means an antibody consisting of As animals other than humans, any animal can be used as long as it can produce a hybridoma such as mouse, rat, mouse, muster, rabbit and the like.
  • cDNAs encoding VH and VL are obtained from hybridomas producing monoclonal antibodies, and inserted into expression vectors for host cells having genes encoding human antibody CH and human antibody CL, respectively.
  • a human chimeric antibody expression vector can be constructed and introduced into a host cell for expression and production.
  • the CH of the human chimeric antibody may be any of those belonging to human immunoglobulin (hereinafter referred to as “hlg”), but is preferably of the MgG class, and further hlg belonging to the MgG class. Any of the subclasses Gl, MgG2, MgG3, MgG4 can be used. As the CL of the human chimeric antibody, any ⁇ class or fly class can be used as long as it belongs to hlg.
  • VH and VL human type homology determining regions (hereinafter referred to as CDRs) of human non-human animal antibodies, amino acids ⁇ ⁇ ⁇ IJ are used for human antibody VH and VL.
  • CDRs VH and VL human type homology determining regions
  • Examples include CDR-grafted antibodies prepared by transplanting to appropriate positions.
  • the CDR-grafted antibody is constructed by constructing cDNA encoding the V region obtained by grafting the VH and VL CDR sequences of the non-human animal antibody to the VH and VL CDR sequences of any human antibody, and the human antibody CH and human antibody.
  • a CDR-grafted antibody expression vector is constructed by inserting each into a host cell expression vector having a gene encoding CL of, and the CDR-grafted antibody is expressed and produced by introducing the expression vector into the host cell. I can do it.
  • the CDR of the CDR-grafted antibody is preferably hlgG class if it belongs to hlg! /, But is also of the hlgG class, and MgGl, MgG2, MgG3, Any of the subclasses such as MgG4 can be used.
  • the CL of the CDR-grafted antibody any ⁇ class or e class can be used as long as it belongs to hlg.
  • Anti-GD2 antibodies Anticancer Res., 13, d31 (1993)], JTLGD3JTL [Cancer Immunol. Immunother., D, 26 0 (1993)], anti-GM2 antibodies [Cancer Res., 54, 1511 (1994)], anti-HER2 antibody [Proc. Natl. Acad. Sci. USA, 89, 4285 (1992)], anti-CD52 antibody [Nature, 332, 323 (1988)], anti MAGE antibody (British J. Cancer, 83, 493
  • Anti-interleukin 6 antibody [Immunol. Rev., 127. 5 (1992)], anti-interleukin 6 receptor antibody [Molecular Immunol., 31 , 371 (1994)], anti-interleukin 5 antibody [Immunol. Rev., 127. 5 (1992)], anti-interleukin 5 receptor antibody, anti-interleukin 4 antibody [Cytokine, 3, 562 (1991)], Anti-interleukin 4 receptor antibody Qj. Immunol • Meth., 2 ⁇ 7, 41 (1998)], anti-tumor necrosis factor antibody [Hybridoma, 13, 183 (199 4)], anti-tumor necrosis factor receptor antibody [Molecular Pharmacol.
  • antibodies that bind to antigens associated with autoimmune diseases include anti-self DNA antibodies [Immu nol. Letters, 72, 61 (2000)], anti-CD1 la antibody, anti-ICAM3 antibody, anti-CD80 antibody, anti-CD2 antibody, anti-CD3 antibody, anti-CD4 antibody, anti-integrin ⁇ 4/3 7 antibody, anti-CD 40L antibody And anti-IL 2 receptor antibody [Immunology Today, 21, 403 (2000)].
  • Anti-gpl20 antibodies [Structure, 8, 385 (2000)], anti-CD4 antibodies. Rheumatology, 25, 2065 (1998)], anti-CCR4 antibodies , Anti-verotoxin antibodies. Clin. Microbiol., 37, 396 (1999)].
  • Examples of the peptide having physiological activity include peptides that maintain the activity of the glycoprotein among the partial fragments of the glycoprotein.
  • a peptide that regulates the enzyme activity or a peptide that retains the structure of the enzyme is also included.
  • the peptide that regulates the activity of the enzyme for example, a peptide that functions as a glycoprotein agonist or antagonist is preferably used. Any peptide may be used as long as it has an activity that enhances the activity of glycoprotein.
  • somatostatin derivative, somatrobin, atrial natriuretic peptide, glucagon, insulin, insulin-like growth factor, Gonadotropin etc. can be raised.
  • the antagonist may be any peptide as long as it has an activity of suppressing the activity of glycoprotein. Specific examples include pegvisomatone.
  • the animal cell used for producing the above-mentioned peptide having physiological activity may be any animal cell as long as the above-mentioned peptide can be produced, but preferably a vector having a gene encoding the peptide to be produced. A transformant into which is introduced is used.
  • a transformed cell into which a vector having a gene encoding a peptide is introduced can be obtained by introducing a recombinant vector containing a DNA encoding a peptide and a promoter into a host cell.
  • the host cell an animal cell having the ability to produce the above substance is used.
  • Any vector can be used as a vector used for preparing the recombinant vector as long as it functions in animal cells used in the present invention.
  • pcDNA I, pcDM8 manufactured by Funakoshi
  • pAGE107 [JP-A-3-22979, Cytotechnology, 3, 133 (1990)]
  • pAS3-3 JP-A-2-227075
  • pcDM8 [Nature, 3 29, 840 (1987) ]
  • PcDNAl / Amp Invitrogen
  • pREP4 Invitrogen
  • PAGE103 U. Biochem. 101, 1307 (1987)]
  • pAGE210 etc.
  • Any promoter can be used as long as it functions in the animal cells used in the present invention.
  • CMV cytomegalovirus
  • SV40 Examples include early promoters, retrovirus promoters, metamouthone promoters, heat shock promoters, SRa promoters, and the like.
  • a human CMV IE gene enhancer or the like may be used together with a promoter.
  • any method for introducing a recombinant vector into a host cell any method can be used as long as it introduces DNA into the cell.
  • the electopore method [Cytot echnology, 3, 133 ( 1990)]
  • calcium phosphate method JP-A-2-227075
  • lipofusion method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987), Virology, 52, 456 (1973)].
  • transformed cells used in the present invention include transformed cells 7-951 (FERM BP-6691) that produce anti-GD3 human chimeric antibodies, and traits that produce anti-CCR4 chimeric antibodies.
  • Transformed cells KM2760 (FERM BP—7054), transformed cells producing anti-CCR4 humanized antibodies KM8759 (FERM BP—8129) and KM8760 (FERM BP—8130), 709 LCA—500D strain (FERM BP—8239), Transformed cells producing anti-IL-5 receptor ⁇ chain chimeric antibody ⁇ 7399 (FERM BP-5649), transformed cells producing anti-IL 5 receptor ⁇ chain human CDR grafted antibody ⁇ 8399 (FERM BP 56 48) And KM9399 (FERM BP—5647), transformed cells producing anti-GM2 human CDR-grafted antibody KM8966 (FERM BP—5105), KM8967 (FERM BP—5106), KM8969 (FERM BP—5527), KM8970
  • any culture method such as batch culture, repeat batch culture, fed-batch culture or perfusion culture may be used, but preferably fed-batch culture is used.
  • the culture volume is usually 10 ⁇ using an Erlenmeyer flask, etc.! Even a small culture volume of OOOmL can be used for commercial production of! ⁇ 200 OOL, usually using a culture tank such as a jar, etc.
  • the culture method used in the present invention may be any method suitable for the animal cells to be used, but usually perfusion culture under conditions of pH 6-8, 30-40 ° C, etc. for 3-20 days. Let's do it for 3-60 days.
  • antibiotics such as streptomycin and penicillin may be added to the medium as needed during the culture.
  • dissolved oxygen concentration control, pH control, temperature control, stirring, etc. can be carried out according to the methods used for normal animal cell culture.
  • animal cells having the ability to produce a substance are cultured in a medium supplemented with a dipeptide, the substance is produced and accumulated in the culture, and the substance is collected from the culture. By doing so, the substance can be produced efficiently.
  • the production method of the present invention includes a direct expression method for producing a peptide in a host cell, a method for secreting and producing a peptide outside a host cell (Molecular Cloning, Second Edition) Etc.
  • Peptides were prepared by the method of Paulson et al. Biol. Chem., 264, 17619 (1989)], the method of Lou et al. [Proc. Natl. Acad. Sci. USA, 86, 8227 (1989), Genes Develop. , 4, 1288 (1990)], or JP-A-5-336963 or WO 94/23021 can be used to actively secrete the cells from the host cell.
  • the desired peptide can be actively secreted outside the host cell by expressing it in such a manner that a signal peptide is bound to the N-terminus of the desired peptide using a genetic recombination technique.
  • the desired peptide produced by the method of the present invention can be isolated and purified using, for example, a normal peptide isolation and purification method.
  • the peptide produced by the method of the present invention is expressed in a dissolved state in the cells, after completion of the culture, the cells are collected by centrifugation, suspended in an aqueous buffer solution, an ultrasonic disrupter, Disrupt the cells using a French press, Manton Gaurin homogenizer, Dynomill, etc. to obtain a cell-free extract.
  • the peptide produced by the method of the present invention is secreted extracellularly, the peptide can be recovered in the culture supernatant. That is, the culture supernatant is obtained by treating the culture by the same method such as centrifugation as described above, and the crude purified sample is obtained from the culture supernatant by using the same isolation and purification method as described above.
  • a purified preparation can be obtained, and the present invention is characterized by adding a dipeptide to an animal cell having an ability to produce a substance and culturing the animal per cell of the substance produced from the cell. It relates to a method for improving productivity. In the present invention, the improvement in productivity per cell of a substance produced by animal cells can be confirmed by determining the specific production rate (SPR) of the substance.
  • SPR specific production rate
  • the specific production rate of a substance refers to the amount of substance produced per cell that produces the substance. Specifically, the amount of substance produced throughout the culture period is compared to the substance that survived during the culture period. By dividing by the number of cells to be produced, the required power S can be obtained.
  • the present invention also relates to a method for maintaining the survival rate of a cell, which comprises culturing by adding a dipeptide to an animal cell having the ability to produce a substance.
  • Cell viability can be determined by collecting cells in the medium over time, measuring the viable cell density, and multiplying the viable cell density by 100.
  • the density of viable cells is determined by a method such as [Culture of Animal Cells: A Manual oi Basic Technique, R. Ian Freshney, Alan R. Liss, Inc., New York (1983)]. You can ask.
  • the present invention also relates to a method for suppressing an increase in the concentration of ammonium ions in a culture solution of a cell, which comprises culturing an animal cell having the ability to produce a substance by adding a dipeptide. .
  • the concentration of ammonium ions in the culture solution can be measured by collecting the culture solution over time and using a potentiometric method such as BioProfile 400 (registered trademark: Nova Biomedical).
  • the present invention relates to a method for suppressing apoptosis of a cell, which comprises culturing by adding a dipeptide to an animal cell having the ability to produce a substance.
  • apoptosis includes early apoptosis.
  • the percentage of early apoptotic cells such as Guava PCA-96 Nexin Kit (manufactured by GE Healthcare Biosciences), is usually present inside the cell membrane, and phosphatidyl that appears outside when an apoptotic reaction occurs.
  • Ms705 / AT III strain (FERM BP 8472) that produces antithrombin, perform batch culture in a 250 mL Erlenmeyer flask and examine the effects of a medium containing L-glutamine and a medium containing L-alanil-L-glutamine. Compared.
  • EX-CELL TM 302 medium As a basic culture medium, a medium (hereinafter referred to as a modified EX-CELL TM 302 medium) in which glutamine was excluded from EX-CELL TM 302 medium (manufactured by JRH Bioscience) was used.
  • methatotrexate hereinafter referred to as MTX
  • MTX methatotrexate
  • L-glutamine 12 mmol / L Wako Pure Chemical Industries
  • L-alanyl-L glutamine 12 mmol / L Malkin
  • Gln culture the culture in which L-glutamine is added to the medium
  • AlGln culture the culture in which L-alanyl-L-glutamine is added to the medium
  • EX-CELL TM 302 medium manufactured by JR Hbioscience
  • MTX 500 nmol / L manufactured by Sigma Aldrich
  • L-glutamin 0 ⁇ 875 g / L (Wako Pure Chemical Industries, Ltd.) Medium
  • Cell suspensions were seeded at 3 ⁇ 10 5 cells / mL in 125 mL, 250 mL, lOOOOmL Erlenmeyer flasks (manufactured by Cojung) at a culture volume of about 10-30%. Thereafter, the cells were cultured at 35 ° C for 4 days, and subcultured several times until the number of cells necessary for seeding of the main culture was obtained.
  • Viable cell density was measured by a dye exclusion method using 0.4% trypan blue solution (Invitrogen).
  • Cumulative viable cell density is shown as the sum of products of viable cell density and elapsed time. The cumulative viable cell density was calculated based on the method shown in Equation 1. [0066] (Formula 1)
  • the medium for expansion until the main culture is EX-CELL TM 302 medium (manufactured by HR Bioscience), MTX 500 nmol / L (manufactured by Sigma Aldrich), and L glutamine 0 ⁇ 875 g / L.
  • the medium added (Wako Pure Chemical Industries, Ltd.) was used. About 10-30% of the medium was placed in a 125 mL, 250 mL, or lOOOOmL Erlenmeyer flask (manufactured by Cojung), and the cell suspension was seeded at 3 ⁇ 10 5 cells / mL.
  • the cells were cultured at 35 ° C for 4 days, and passaged several times until the number of cells required for seeding of the main culture was obtained.
  • the cells were seeded at 3 X 10 5 cells / mL in a 2 L bioreactor (Able) filled with 600 mL of the basic medium described below.
  • the basic culture medium is EX-CELL TM 302 medium (manufactured by GM Bioscience), modified medium with glutamine removed, MTX 500 nmol / L (Sigma Al A medium supplemented with L. glutamine 1.75 g / L (Wako Pure Chemical Industries) was used.
  • the feed medium contains amino acids [(L-alanine 0 ⁇ 14 g / L, L-arginine monohydrochloride 0 ⁇ 47 g / L, L-parasine monohydrate 0 ⁇ 16 g / L, L-cystine dihydrochloride 0 ⁇ 51 g / L, L— Glutamic acid 0.42 g / L, L Histidine monohydrochloride dihydrate 0.24 g / L, L Iso-Isine 0.
  • Gln-G In culture The culture in which L-glutamine is added to the basic medium and the feed medium is referred to as “Gln-G In culture”, and the culture in which L-alanil-L-glutamine is added to the feed medium in the basic medium and the feed medium is referred to as “Gln-AlaGln culture”. And).
  • the viable cell density was measured by the same method as in Example 1. In this example, the viable cell density was measured every day from the beginning of the culture to the 14th day, and the cumulative viable cell density was calculated from Equation 1 described in Example 1 using the measured viable cell density.
  • the protein concentration was measured by HPLC.
  • PA ID Sensor Cartridge (Applied Systems) was used as the column, and L-7400 (detection wavelength 280 nm, manufactured by Hitachi, Ltd.) was used as the detector.
  • Ammonium ion concentration is BioProfile 400 (registered trademark: Nova Biomedical)
  • SPR which represents the amount of protein produced by a unit cell per unit time, was calculated from the following equation 2 using the measured protein concentration and the calculated cumulative viable cell density.
  • SPR g / 10 6 cells / day protein concentration (mg / L) ⁇ cumulative viable cell density (10 6 cells / mL X days)
  • the survival rate was 90% or more from the start of culture to the ninth day of culture.
  • the cell viability in Gin-Gin culture on the 10th day of culture was 59%, while that in Gin-AlaGln culture was as high as 95%.
  • the maximum reached viable cell density was 1.0 ⁇ 10 7 cells / mL or more in the culture using any of the feed media.
  • the cumulative viable cell density was approximately equal to 9.7 X 10 7 cells / mL X day for Gin-Gin culture and 9.3 X 10 7 cells / mL X day for Gin-AlaGln culture.
  • the ammonium ion concentration decreased to 311111101 / in the 011-8, 1 & 011 culture, compared to about 6 mmol / L in the Gin-Gin culture. It was revealed that the ammonium ion concentration in the medium was reduced when the feed medium supplemented with Ranil L-glutamine was used.
  • the protein concentration increased to 131% in Gln-AlaGln culture, assuming that the value in Gin-Gin culture was 100%.
  • SPR increased to 121% in Gin-AlaGln culture, assuming the value in G1 n-Gin culture as 100%.
  • the culture using the feed medium supplemented with L-alanil-L-glutamine produced antithrombin while maintaining the viability of the production cells as compared with the culture using the feed medium supplemented with glutamine. It became clear that productivity per cell was improved.
  • the medium for expansion until the main culture is EX-CELL TM 302 medium (manufactured by HR Bioscience), MTX 500 nmol / L (manufactured by Sigma Aldrich), L glutamine 1 ⁇ 75 g / L A medium supplemented with (Wako Pure Chemical Industries, Ltd.) was used. About 20% of the medium was placed in a 125 mL, 250 mL, 500 mL or lOOOOmL volume Erlenmeyer flask (manufactured by Cojung), and the cell suspension was seeded at 3 ⁇ 10 5 cells / mL. The cells were cultured at 35 ° C for 4 days, and subcultured several times until the number of cells required for seeding of the main culture was obtained.
  • the cells were seeded at 3 X 10 5 cells / mL in a 250 mL Erlenmeyer flask (manufactured by Cojung) filled with 40 mL of the following basic medium. Thereafter, the cells were cultured at 35 ° C, 100 rpm, pH 7.1 for 14 days.
  • the modified EX—CELL TM 302 medium used in Example 2 was added to MTX 500 nmol / L (Sigma Aldrich) and L-glutamine 0 ⁇ 87 g / L (Wako Pure Chemical Industries) or A medium supplemented with L-alanil-L-glutamine 1 ⁇ 30 g / L (Kyowa Hakko Kogyo Co., Ltd.) was used.
  • the feed medium contained amino acids [L alanine 0 ⁇ 14 g / L, L arginine monohydrochloride 0 ⁇ 47 g / L, L-rasparagine monohydrate 0 ⁇ 16 g / L, L—cystine dihydrochloride 0 ⁇ 51 g / L, L—glutamic acid 0.42 g / L, L histidine monohydrochloride dihydrate 0.24 g / L, L-Isoguchiine 0.59 g / L, L-leucine 0.59 g / L, L-lysine monohydrochloride 0.82 g /: L, L phenyl lanine 0.37 g / L, L proline 0.22 g / L, L-serine 0.
  • the culture solution is collected on days 1, 3, 4, 7, 10, 12, and 14 and the viable cell density (cells / mL), protein concentration (mg / L), and ammonium ion concentration (mmol / L) are measured. It was measured.
  • the viable cell density was measured by the same method as in Example 1.
  • the protein concentration and the ammonium concentration were measured in the same manner as in Example 2.
  • the cumulative viable cell density (cells / mL X day) and SPR g / 10 6 cells / day were calculated by the following method.
  • the cumulative viable cell density was calculated from Equation 1 described in Example 1 using the measured viable cell density.
  • SPR is the formula described in Example 2 using the measured protein concentration and the calculated cumulative viable cell density.
  • the results are shown in Figs.
  • the survival rate showed a good value of 95% or more from the start of culture to the seventh day of culture in any culture.
  • the maximum viable cell density showed good growth in all cultures exceeding 4.5 ⁇ 10 6 cells / mL.
  • the concentration of ammonium ions at the time of incubation was 12. lmmol / L in Gin-Gin culture, whereas it was 7.4 mmol / L in Gln-AlaGln culture, and 5.2 mmol in AlaGln-AlaGln culture. / L, and the addition of L-alanil-L-glutamine to the medium reduced the ammonia concentration.
  • the cumulative viable cell density for 14 days is 2.6 X 10 7 cells / mL X day for Gin—Gin culture, 3 ⁇ 9 X 10 7 cells / mL X day for Gin—AlaGln culture, 3 ⁇ for AlaGln—Ala Gin culture OX 10 7 cells / mL X day, the highest value was obtained when L-alanil-L-glutamine was added only to the feed medium.
  • the protein concentration increased to 180% in the Gin-AlaGln culture and 194% in the AlaGln-AlaGln culture with the addition of L-alanil-L-glutamine, assuming that the value in the Gin-Gin culture was 100%.
  • SPR increased to 120% for Gin-AlaGln culture and 167% for AlaGln-AlaGln culture with the addition of L-alanil-L-glutamine, assuming a value of 100% for Gin-Gin culture.
  • the medium for expansion until the main culture includes EX-CELL TM 302 medium (manufactured by HR Bioscience), MTX 500 nmol / L (manufactured by Sigma Aldrich), L-glutamine 1. 75 g / L A medium supplemented with (Wako Pure Chemical Industries, Ltd.) was used. About 20% of the medium was placed in a 125 mL, 250 mL, 500 mL, or lOOOOmL Erlenmeyer flask (manufactured by Cojung), and the cell suspension was seeded at 3 ⁇ 10 5 cells / mL. The cells were cultured at 35 ° C for 4 days, and subcultured several times until the number of cells required for seeding of the main culture was obtained.
  • the cells were seeded at 3 X 10 5 cells / mL in a 2 L volume bioreactor (Able) filled with 600 mL of the basic medium described below.
  • the modified EX—CELL TM 302 medium used in Example 2 MT X500 nmol / L (Sigma Aldrich) and L-glutamine 0 ⁇ 87 g / L (Wako Pure Chemical Industries) or A medium supplemented with L-alanil-L-glutamine 1 ⁇ 30 g / L (Kyowa Hakko Kogyo Co., Ltd.) was used.
  • the feed medium contains amino acids [L-alanine 0 ⁇ 14 g / L, L-arginine monohydrochloride 0 ⁇ 47 g / L, L-parasine monohydrate 0 ⁇ 16 g / L, L-cystine dihydrochloride 0 ⁇ 51 g / L L, L--Glutamic acid 0.42 g / L, L histidine monohydrochloride dihydrate 0.24 g / L, L iso-icine 0.59 g / L, L leucine 0.59 g / L, L lysine monohydrochloride 0.
  • the culture solution is collected every day, and the viable cell density (cells / mL), protein concentration (mg / U, ammonium ion concentration (mmol / L), L glutamine concentration, and L-alanil-L-glutamine Concentration (mmol / L) was measured.
  • the viable cell density was measured by the same method as in Example 1.
  • the protein concentration and ammonium ion concentration were measured in the same manner as in Example 2.
  • L-glutamine concentration and L-alurul-L-glutamine concentration were measured by inducing the collected culture medium with FMOC-C1 according to the following procedure.
  • 270 ⁇ L of lOOmmol / L borate buffer prepared from boric acid (manufactured by Kanto Chemical Co., Ltd.) and sodium hydroxide (manufactured by Kokusan Kagaku Co., Ltd., ⁇ 9.0) and acetone (Kokusan Chemical Co., Ltd.)
  • the product was mixed with 300 L of a 1.5 mg / mL FMOC-C1 (manufactured by Tokyo Kasei Co.) solution.
  • the cumulative viable cell density was calculated from Equation 1 described in Example 1 using the measured viable cell density.
  • SPR was calculated from Equation 2 described in Example 2 using the measured protein concentration and the calculated cumulative viable cell density.
  • Guava EasyCyte Plus (manufactured by GE Healthcare Bioscience) was used as a device for measuring the ratio of early apoptotic cells.
  • Guava PCA-96 Nexin Kit (manufactured by GE Healthcare Bioscience) was used as the measurement reagent, and the measurement was performed according to the experimental procedure attached to the measurement reagent.
  • Gin-AlaGln culture decreased to 3.4 mmol / L on the 8th day, indicating that the addition of L-alanyl L-glutamine decreased the ammonium ion concentration.
  • the protein concentration increased to 109% in the Gin-AlaGln culture, assuming that the Gin-Gin culture value was 100%.
  • SPR increased to 10 6% for Gin-AlaGln culture and 115% for AlaGln-AlaGln culture with the addition of L-alanyl-L-glutamine, assuming the value of Gin-Gin culture as 100%.
  • the concentration of L-alanil-L-gnoretamine was OmM force S until the third day of culture, and then the concentration increased each time the feed medium was added until the eighth day of culture. After the 11th day of culture, the concentration of L-farnil and L-glutamine began to decrease and reached approximately OmM on the 14th day.
  • the proportion of cells with early apoptosis was 7.3% in Gin-AlaGln culture and 4.1% in AlaGln-AlaGln culture, compared to 7.3% in Gin-Gin culture on day 8 of culture.
  • the percentage of early apoptotic cells was suppressed by the addition of L-alanil-L-glutamine.
  • the Gin-Gin culture was 11.5%, while the Gln-AlaGln culture was 6.7% and the AlaGln-AlaGln culture was 3.6%.
  • Gin-Gin culture was 14.1%, Gln-AlaGln culture was 14.8%, and AlaGln-AlaGln culture was 8.3%.
  • the medium for expansion until the main culture is EX-CELL TM 302 medium (manufactured by JRH Bioscience), MTX 500 nmol / L (manufactured by Sigma Aldrich), L glutamine (manufactured by Wako Pure Chemical Industries, Ltd.) 1.
  • a medium supplemented with 75 g / L was used.
  • a cell suspension was seeded at 3 ⁇ 10 5 cells / mL.
  • the cells were cultured at 35 ° C for 4 days, and subcultured several times until the number of cells required for seeding of the main culture was obtained.
  • the feed medium contained amino acids [L-alanine 0 ⁇ 14 g / L, L-arginine monohydrochloride 0 ⁇ 47 g / L, L-parasine monohydrate 0 ⁇ 16 g / L, L-cystine dihydrochloride 0 ⁇ 51 g / L L, L--Glutamic acid 0.42 g / L, L histidine monohydrochloride dihydrate 0.24 g / L, L iso-icine 0.59 g / L, L leucine 0.59 g / L, L lysine monohydrochloride 0.
  • 022g / L Folic acid 0.02 2g /, myo Inosinole 0.040g /, Niacinamide ⁇ . 022g /, Pyrido, xalhydrochloric acid 0.022g / L, Riboflavin 0.0021g / L, Thiamine hydrochloride 0 022g / L, Shianocobalamin 0.073mg / L (sold by Sigma-Aldrich)], Recombinant Human Insulin 0 ⁇ 31g / L (GJ Bioscience), Ethanolamine 0 ⁇ 0 25g / L (manufactured by Sigma-Aldrich), 2 mercaptoethanol 0.009g / L (manufactured by Sigma-Aldrich), soy hydrolyzate HY—SOY8g / L (manufactured by Quest International), sodium selenite 16.
  • the culture solution was collected on days 4, 7, 9, 11, 13, and 14 of the culture, and the viable cell density (cell / mU and protein concentration (mg / L) was measured.
  • the viable cell density was measured by the same method as in Example 1.
  • the protein concentration was measured in the same manner as in Example 2.
  • the cumulative viable cell density (cells / mL X day) and SPR g / 10 6 cells / day were calculated by the following method.
  • the cumulative viable cell density was calculated from Equation 1 described in Example 1 using the measured viable cell density.
  • SPR was calculated from Equation 2 described in Example 2 using the measured protein concentration and the calculated cumulative viable cell density.
  • the medium for expansion until the main culture is EX-CELL TM 302 medium (GLA).
  • GLA EX-CELL TM 302 medium
  • MTX500 nmol / L Sigma-Aldrich
  • L-glutamine 1.75 g / L Wang-OOOmL Erlenmeyer flask (manufactured by Cojung)
  • the cells were cultured at 35 ° C for 4 days, and subcultured several times until the number of cells required for seeding of the main culture was obtained.
  • the feed medium contained amino acids [L-alanine 0 ⁇ 14 g / L, L-arginine monohydrochloride 0 ⁇ 47 g / L, L-parasine monohydrate 0 ⁇ 16 g / L, L-cystine dihydrochloride 0 ⁇ 51 g / L L, L--Glutamic acid 0.42 g / L, L histidine monohydrochloride dihydrate 0.24 g / L, L iso-icine 0.59 g / L, L leucine 0.59 g / L, L lysine monohydrochloride 0.
  • 022g / L Folic acid 0.02 2g /, myo Inosinole 0.040g /, Niacinamide ⁇ . 022g /, Pyrido, xalhydrochloric acid 0.022g / L, Riboflavin 0.0021g / L, Thiamine hydrochloride 0 022g / L, Shianocobalamin 0.073mg / L (sold by Sigma-Aldrich)], Recombinant Human Insulin 0 ⁇ 31g / L (GJ Bioscience), Ethanolamine 0 ⁇ 0 25g / L (manufactured by Sigma-Aldrich), 2 mercaptoethanol 0.009 g / L (Sigma Soy hydrolyzate HY—SOY8g / L (Quwest International), Sodium selenite 16.8 g / L (Sigma—Aldrich), Cholesterol lipid concentrated solution 2mL / L (250 X aqueous solution, manufactured by
  • a feed medium of 6.25% of the initial medium amount was added on the 47th and 9th days from the start of the culture.
  • the viable cell density was measured in the same manner as in Example 1.
  • the specific growth rate (h- 1 ) was calculated from the following formula 3 using the viable cell density on the 4th day of culture /
  • h— (Natural logarithm of viable cell density on day 4)
  • the results of the seeded live cell density are shown in Fig. 9 and Fig. 10.
  • the specific growth rate is 0 in the culture without ammonium chloride. 0187h— 0 for lOmmol / caroculture In culture of 20 mmol / L supplemented carodium, 0.0109 h—in culture of 30 mmol / L supplemented caroten, 0 0072 h—in culture of supplemented 40 mmol / L, 0.4 0041 h—in culture supplemented with 50 mmol / L—0.0025 h— 1 And 60 mMol / L addition, it was 0.0033h- 1 , indicating that the specific growth rate decreased as the concentration of ammonium chloride in the basic medium increased.
  • Anti-CD20 antibody production by 2L bioreactor Effect of ammonium ions in the culture medium in the cultivation of CHO cell line Ms704 / CD20
  • the medium for expansion until the main culture is EX-CELL 1 M 302 medium (manufactured by HR Bioscience), MTX 500 nmol / L (Sigma-Aldrich) and L glutamine 1 ⁇ 75 g A medium supplemented with / L (manufactured by Wako Pure Chemical Industries, Ltd.) was used.
  • the feed medium contained amino acids [L-alanine 0 ⁇ 14 g / L, L-arginine monohydrochloride 0 ⁇ 47 g / L, L-parasine monohydrate 0 ⁇ 16 g / L, L-cystine dihydrochloride 0 ⁇ 51 g / L L, L--Glutamic acid 0.42 g / L, L histidine monohydrochloride dihydrate 0.24 g / L, L iso-icine 0.59 g / L, L leucine 0.59 g / L, L lysine monohydrochloride 0.
  • 022g / L Folic acid 0.02 2g /, myo Inosinole 0.040g /, Niacinamide ⁇ . 022g /, Pyrido, xalhydrochloric acid 0.022g / L, Riboflavin 0.0021g / L, Thiamine hydrochloride 0 022g / L, Shianocobalamin 0.073mg / L (sold by Sigma-Aldrich)], Recombinant Human Insulin 0 ⁇ 31g / L (GJ Bioscience), Ethanolamine 0 ⁇ 0 25g / L (manufactured by Sigma-Aldrich), 2 mercaptoethanol 0.009 g / L (Sigma Mar Aldrich), soy hydrolyzate HY— SOY8g / L (Quwest International), sodium selenite 16.8 g / L (Sigma Aldrich), cholesterol concentrated solution 2mL / L (250 X aqueous solution, manufactured by Invitrog
  • the culture broth was collected every day, and the viable cell density (cells / mL), protein concentration (mg / L), and ammonium ion concentration (mmol / L) were measured.
  • the viable cell density was measured by the same method as in Example 1.
  • the cumulative viable cell density (cells / mL X day) was calculated by the following method.
  • the cumulative viable cell density was calculated from Equation 1 described in Example 1 using the measured viable cell density.
  • the results are shown in Fig. 11.
  • the survival rate on the 5th day of culture decreased to 92% or less in the culture supplemented with ammonium chloride.
  • the culture without ammonium chloride it was maintained at 98% or more.
  • the maximum reached viable cell density was over 5.0 ⁇ 10 6 cells / mL in the culture supplemented with ammonium chloride.
  • culture without ammonium chloride showed a good growth of 1.1 X 10 7 cells / mL.
  • the protein concentration decreased to 61% in the culture without ammonium chloride, assuming that the value was 100% in the culture without ammonium chloride.
  • a dipeptide is added in the method for culturing animal cells having the ability to produce a substance.
  • a method for improving the per-cell productivity of a substance produced from the cell characterized by culturing, characterized by adding a dipeptide to an animal cell having the ability to produce the substance, and culturing.
  • a method for maintaining the viability of the cells a method for culturing animal cells having the ability to produce substances, and culturing with the addition of a dipeptide. It is possible to provide a method for suppressing an increase in the ON concentration, and a method for suppressing apoptosis of the cell, which comprises culturing by adding a dipeptide to an animal cell having the ability to produce a substance.

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Abstract

La présente invention concerne : un procédé de culture de cellules animales capables de produire une substance caractérisé en ce que, dans le procédé de culture de cellules animales capables de produire la substance, la culture est réalisée par ajout d'un dipeptide ; un procédé de production d'une substance caractérisé en ce que, dans le procédé de production de la substance par culture de cellules animales capables de produire la substance, la culture est réalisée par ajout d'un dipeptide et la substance ainsi formée et accumulée dans le milieu de culture est récoltée de la culture ; un procédé d'augmentation de la productivité par cellule d'une substance produite par des cellules animales capables de produire la substance caractérisé en ce que les cellules sont cultivées par ajout d'un dipeptide ; un procédé de maintien du taux de survie de cellules animales capables de produire une substance caractérisé en ce que les cellules sont cultivées par ajout d'un dipeptide ; un procédé d'inhibition d'une augmentation de la concentration en ion ammonium d'un milieu de culture liquide de cellules animales capables de produire une substance caractérisé en ce que les cellules sont cultivées par ajout d'un dipeptide ; et un procédé d'inhibition de l'apoptose de cellules animales capables de produire une substance caractérisé en ce que les cellules sont cultivées par ajout d'un dipeptide.
PCT/JP2007/067941 2006-09-20 2007-09-14 Procédé de production d'une substance WO2008035631A1 (fr)

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CN102892878A (zh) * 2010-04-23 2013-01-23 生命技术公司 包含小肽的细胞培养基
CN103080300A (zh) * 2010-08-05 2013-05-01 安姆根有限公司 增加细胞培养物的产率和活力的二肽
CN112041430A (zh) * 2017-12-08 2020-12-04 朱诺治疗学股份有限公司 用于培养细胞的无血清培养基配制品及其使用方法
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