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WO2007049567A1 - Method for production of substance - Google Patents

Method for production of substance Download PDF

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Publication number
WO2007049567A1
WO2007049567A1 PCT/JP2006/321082 JP2006321082W WO2007049567A1 WO 2007049567 A1 WO2007049567 A1 WO 2007049567A1 JP 2006321082 W JP2006321082 W JP 2006321082W WO 2007049567 A1 WO2007049567 A1 WO 2007049567A1
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WO
WIPO (PCT)
Prior art keywords
cell
culture
cells
substance
antibody
Prior art date
Application number
PCT/JP2006/321082
Other languages
French (fr)
Japanese (ja)
Inventor
Yoshinobu Konno
Naoto Sakai
Kentaro Sakai
Ryuma Nagano
Masamichi Koike
Shinji Hosoi
Masakazu Takagishi
Yutaka Makimoto
Original Assignee
Kyowa Hakko Kogyo Co., Ltd.
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.)
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Publication date
Application filed by Kyowa Hakko Kogyo Co., Ltd. filed Critical Kyowa Hakko Kogyo Co., Ltd.
Publication of WO2007049567A1 publication Critical patent/WO2007049567A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/005—Glycopeptides, glycoproteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

  • the present invention is characterized by culturing animal cells in a medium to which a substance selected from lignans, flavonoids, histone deacetylase inhibitors, terpenoids, kojic acid or derivatives thereof is added. And a medium supplemented with a substance selected for the substance strength selected from lignans, flavonoids, histone deacetylase inhibitors, terpenoids, kojic acid or derivatives thereof.
  • the present invention relates to a method for improving the productivity of a substance per cell.
  • Peptides have various pharmaceutical uses.
  • peptides with immune functions such as antibodies are used to reduce rejection of renal transplantation, antiviral agents against RSV childhood infection, anticancer agents against breast cancer, etc.
  • antibodies are expected to become increasingly important as pharmaceuticals.
  • a recombinant cell into which the gene encoding the glycoprotein has been introduced is cultured, and the recombinant protein produced in the culture is produced.
  • the recombinant protein produced in the culture is produced.
  • genetically engineered glycoproteins are produced only in animal cells as fully mature forms, so many genetically modified glycoproteins are produced using animal cells. It has been.
  • Non-patent Document 1 Non-patent Document 1
  • Non-patent Document 2 Animal cell culture in such a large culture tank is a cell culture method. There is a problem such as insufficient supply of oxygen to the water or an increase in dissolved carbon dioxide in the medium.
  • by-products such as lactic acid and ammonia produced by animal cells in culture show inhibition of animal cell growth
  • glutamine synthetase that can grow on a glutamine-free medium that is a source of ammonia Cells into which a gene has been introduced (Patent Document 1) are used for the production of pharmaceuticals, and such a device has been effective mainly by extending culture.
  • Non-Patent Document 3 In order to improve the productivity of substances such as peptides per cell using mammalian cells such as CHO cells or myeloma cells often used for peptide production, expression vector systems, cell selection methods, culture methods, purification methods (Non-Patent Document 3).
  • the osmotic pressure of mammalian cell culture medium is generally about 260-320 mOsm / kg. It is known that the productivity per substance of cells improves when the osmotic pressure is high, such as 450-600 mOsmZkg. (Patent Literature 3, Patent Literature 4).
  • a method for improving the productivity of a substance such as a peptide using mammalian cells such as CHO cells or myeloma cells a method of adding various substances that improve productivity to the medium is known. Yes. Specifically, a method of culturing by adding substances such as butyric acid (Patent Document 5), retinoic acid (Patent Document 6), and Coenzyme Q10 (Patent Document 7) is known.
  • Noreproic acid and its derivatives are inhibitors of histone deacetylase, and are known as anti-epileptics (Patent Document 8).
  • Sesamol is known as a sugar-lipid metabolism improving agent (Patent Document 9).
  • Flavones such as luteolin and acacetine are known as compounds aimed at treating infectious diseases (Patent Document 10).
  • Isoflavones such as daidzein are known to be incorporated into topical skin preparations as female hormone production promoters! / Speak (Patent Document 11). It is known that ondisaponin and glycyrrhetinic acid are also added to the external preparation for skin (Patent Documents 12 and 13). However, it is well known that any substance has an effect on the per-cell productivity of animal cells when added to the medium and cultured in animal cells.
  • camphor is known to be used as a topical preparation for preventing induced stimulants and corneal disorders (Non-patent Document 4, Patent Document 14). It is known that when camphor is added to a medium, it has an influence on the proliferation of conflicting cells such as growth inhibition (Non-patent document 5) and growth promotion (Non-patent document 6). While working hard, It is well known how animal cells are cultivated by adding them to the medium, which has an effect on the productivity of the substance per cell.
  • Patent Document 15 Kojic acid or its derivatives are known as whitening agents.
  • Patent Document 16 the addition of kojic acid to the medium for the purpose of examining the chelating effect on transferrin added to the chemically synthesized medium is known.
  • the addition of kojic acid has been reported to inhibit the growth of CHO cells, but there is no description of the effect of kojic acid on the per-cell productivity of the substance.
  • animal cells are cultured with the derivative added to the medium, it is not known what effect the substance has on the productivity per cell.
  • Patent Document 1 US5747308
  • Patent Document 2 WO01 / 29246
  • Patent Document 3 W096Z39488
  • Patent Document 4 US4724206
  • Patent Document 5 JP-A-8-9968
  • Patent Document 6 US5155136
  • Patent Document 7 WO03 / 046174
  • Patent Document 8 WO02 / 007722
  • Patent Document 9 JP-A-11 246427
  • Patent Document 10 WO0lZ003681
  • Patent Document 11 Japanese Unexamined Patent Application Publication No. 2004-67590
  • Patent Document 12 JP-A-8-133948
  • Patent Document 13 Japanese Patent Laid-Open No. 2003-160463
  • Patent Document 14 JP 2004-339119 A
  • Patent Document 15 JP-A-5-310727
  • Patent Document 16 US6767741
  • Non-Patent Document 1 Nature Rev. Drug. Discov., 3, 383 (2004)
  • Non-Patent Document 2 Nature biotechnology, 19, Mar. 21, 184 (2001)
  • Non-Patent Document 3 Nature biotechnology, 22, Nov. 11, 1393 (2004)
  • Non-Patent Document 4 Pharmacological Journal of Japan, 83, 207 (1984)
  • Non-Patent Document 5 Dentistry, 75 (5), 985 (1987)
  • Non-Patent Document 6 Dental Medicine, 52 (6), 745 (1989)
  • a method for improving the productivity of a substance to be produced is required in addition to a method for culturing animal cells and producing the cellular force substance.
  • the present invention relates to the following (1) to (27).
  • An animal cell having the ability to produce a substance is cultured in a medium supplemented with at least one substance selected from the following (a) to (e) force: To improve the per-cell productivity of a certain substance.
  • animal cells in a medium to which at least one substance selected from lignans, flavonoids, histone deacetylase inhibitors, terpenoids, camphor, kojic acid or their derivatives are added.
  • a method for producing a substance characterized by culturing lydanan, flavonoids, histone deacetylase inhibitor, terpenoids, camphor, kojic acid or a derivative thereof, at least one substance selected from is provided a method for improving the productivity per cell of a substance, characterized by using an added medium.
  • Fig. 1 shows the specific production rate per cell (pgZ cell Z day) on the 14th day from the start of the culture when the Ms704-CD20 strain was used in a batch flask in an Erlenmeyer flask.
  • Figure 2 shows the specific production rate per cell (pgZ cell Z day) on the 14th day from the start of the culture when fed batch culture was performed in an Erlenmeyer flask using Ms705-pKAN-— strain.
  • FIG. 3 shows the concentration of AT- ⁇ ⁇ ⁇ ⁇ produced against the cumulative viable cell density (cells / mL x day) when fed-batch culture was performed in Erlenmeyer flasks using Ms705-pKAN- ⁇ strain. It is the figure which showed (mg / L).
  • X indicates control culture
  • mouth indicates 0.12 mmol ZL
  • ⁇ indicates 0.23 mmol / L
  • ⁇ indicates 0.39 mmol / L
  • fist indicates 0.55 mmol / L with camphor.
  • FIG. 4 A graph showing the specific production rate per cell (pgZ cell Z day) when Ms704ZCD20 strain was used in a Erlenmeyer flask!
  • China indicates culture with camphor and ⁇ indicates control culture.
  • FIG. 5 is a graph showing the specific production rate per cell (pgZ cell Z days) when a fed batch culture was performed in an Erlenmeyer flask using the 709 LCA-500G strain.
  • China represents the culture with the addition of camphor
  • ⁇ represents the control culture.
  • FIG. 6 is a graph showing changes in the density of viable cells with time when! /, And Fed batch culture were performed on Erlenmeyer flasks using Ms704ZCD20 strain.
  • ⁇ indicates the culture with the addition of camphor at the start of the culture
  • ⁇ indicates the culture with the addition of camphor on the third day from the start of culture
  • Kuni indicates the culture with the addition of camphor on the fifth day from the start of the culture.
  • X adds camphor on the 9th day after the start of culture!
  • ⁇ indicates a control culture.
  • FIG. 7 is a graph showing the specific production rate per cell (pgZ cell Z days) when! /, Fed-batch culture was performed in an Erlenmeyer flask using Ms704ZCD20 strain.
  • ⁇ indicates the culture at each time when the country camphor was added, and ⁇ indicates the control culture.
  • FIG. 8 is a graph showing 15 days from the start of culture when 709 LCA-500G strain was fed batch culture in an Erlenmeyer flask and supplemented with kojic acid at a concentration of ImmolZL.
  • FIG. 3 is a graph showing a change with time in the density of living cells. In the figure, (b) indicates the culture added with kojic acid at the same time as the start of cultivation.
  • Fig. 9 shows the specific production rate (pgZ cell Z day) per cell when fed batch culture was performed in an Erlenmeyer flask using the 709 LCA-500G strain.
  • the frame in the figure shows the left force control, the culture with addition of kojic acid on the 3rd and 5th days at the same time as the start of the culture.
  • the white column represents the culture in which kojic acid was added at 0.5 mmol ZL and the black column was added with kojic acid at 1. O mmol ZL.
  • the present invention cultivates animal cells in a medium to which at least one substance selected also for lignans, flavonoids, histone deacetylase inhibitors, terpenoids, kojic acid or derivatives thereof is added. And in a medium containing lignans, flavonoids, histone deacetylase inhibitors, terpenoids, kojic acid, or at least one substance whose inductive capacity is also selected. It is related with the method of improving the productivity per cell of a substance characterized by cultivating an animal cell in (3).
  • the lignans, flavonoids, histone deacetylase enzyme inhibitors and terpenoids added to the medium in the present invention also include analogs and derivatives such as glycosides of these substances.
  • Lignans are a group of compounds whose basic structure is [C 6 -C 3 ], in which two molecules of phenylpropanoid are bonded between ⁇ -carbons. ) Neligignan bonded between carbons other than 8 carbons and oxygen, sesquilignan bonded with 3 molecule propanoids, dilignan bonded with 4 molecules, etc. are also included in the lignans.
  • the lignans used in the present invention are not particularly limited as long as they are lignans.
  • podophyllotoxin sesamin, sesaminol, sesamonole, sesamorinol, sesamolin, cisandrin, trashin A, magnolol, Examples include honokiol, phenolpropanoid, and trashin A. Sesamol is preferably used.
  • Flavonoids are a general term for a mixture of pigment components and polyphenols widely contained in plants.
  • the basic structure is [C 6 -C 3 _C 6 ] in which two phenyl groups are bonded via three carbons. Yes Refers to a group of compounds.
  • the flavonoids used in the present invention are not particularly limited, but specifically, flavanone, flavone, chalcone, flavanol (catechin), flavonol, flavonol, aurone, flavan-3,4-diol (leucoanthocyan), isoflavone, Examples include isoflavonoids, and flavones and isoflavonoids are preferably used.
  • flavones strong luteolin, acacetine, and the like, which are exemplified by noreteolin, acacetin, chrysin or apigenin, are preferably used.
  • isoflavonoid force daidzein such as daidzein, genistin or glycitin is preferably used.
  • Examples of the histone deacetylase inhibitor used in the present invention include valproic acid and valproic acid.
  • Sodium valproate which includes sodium proate, sodium butyrate, sveroidalide, avicidin trapoxin, hydroxamic acid and the like, is preferably used.
  • Terpenoids are isoprene-polymerized compounds. Terpenoids include monoterpenes (C), sesquiterpenes (C), diterpenes (C), sesterterpenes (C), trite
  • Monoterpenes include gala-ol, nerol, linalool, citral (gera-al), cineol, citronellol, menthol, limonene, terbinerol, force norebon, nonon, gyon, camphor, borneol, anetor, eugenol. It is done.
  • sesquiterpenes include fuarnesol, nerolidol, juvenile hormone, humulene, cariophyllene, elemental, casinodal, kadinene, and chin.
  • Examples of diterpenes include gerageruger-ol, phytol, abietic acid, pimaragen, daphnetoxin, taquinol, and saffirne.
  • Triterpenes include squalene, limonin, cameliagenin, hopane, lanosterol, saponin and the like.
  • Examples of tetraterpenes include carotenoids.
  • Examples of glycosides include glycyrrhetinic acid and its derivatives.
  • the terpenoid used in the present invention is not particularly limited as long as it is a terpenoid, but camphor, saponin, glycyrrhetinic acid and derivatives thereof are preferably used.
  • Onjisaponin G is an example of saponin.
  • Daricylretinoic acid and its derivatives include OC glycyrrhetinic acid, ⁇ -glycyrrhetinic acid, a stearyl glycyrrhetinate, 13 stearyl glycyrrhetinate, pyridoxine glycyrrhetinate, j8-glycyrrhetinic acid pyridoxine, a glycerin glycyrrhetinate, glycerin 13 glycyrrhetinate, Glycyrrhetinic acid derivatives such as 3-succinoxy glycyrrhetinic acid ninatrium, 18 ⁇ -glycyrrhizic acid, 18 j8-glycyrrhizic acid, 18 ⁇ -glycyrrhizic acid methyl ester, 18
  • camphor in addition to camphor, also referred to as camphor, camphor, camphor, etc.
  • camphor and its analogs may be either d-form, 1-form, dl-form optical isomers or camphor derivatives.
  • substances containing these, such as essential oils, can also be used.
  • camphor derivatives include camphanic acid and its salts, camphene, succinic acid and its salts, camphorquinone, camphorquinone 3-oxime, camphorsulfoxide, 10-strong sulfo-loxaziridine, camphorsulfone Examples include acids and salts thereof, camphor t-tosylhydrazone, or ketopic acids and salts thereof.
  • glucuronic acid conjugates of these derivatives metabolites such as 3-hydroxycamphor, 5-hydroxycamphor, 8-hydroxy, 9-hydroxycamphor, and the like can be mentioned.
  • Kojic acid to be added to the medium in the present invention is a compound represented by the following structural formula.
  • Examples of kojic acid used in the present invention include the following compounds, salts and salts of the following compounds. Also includes saccharides.
  • the salt examples include metal salts such as alkali metal salts (for example, sodium salts, potassium salts, and cesium salts), alkaline earth metal salts (for example, calcium salts, magnesium salts, and the like), ammonia -Um salt, salt with organic base (for example, trimethylamine salt, triethylamine salt, pyridine salt, picoline salt, ethano-lamine salt, trihetano-lamine salt, dioxyhexylamine salt, N, N, Benzylethylenediamine salt), salts with amino acids ( For example, arginine salt, aspartate, glutamate and the like).
  • alkali metal salts for example, sodium salts, potassium salts, and cesium salts
  • alkaline earth metal salts for example, calcium salts, magnesium salts, and the like
  • ammonia -Um salt salt with organic base (for example, trimethylamine salt, triethylamine salt, pyridine salt, picoline salt, e
  • glycosides include glucose-bound glycosides, kojic acid monodalcoside, kojic acid tridarcoside, kojic acid tridarcoside (Japanese Patent Laid-Open No. 07-2336496) and Kojic acid 5-0-a-D-Dalcobilanoside (JP 05-078383), fructose-bound kojic acid fructosides (JP 05-0778383, JP 10-099091), galactose-bound kojic acid galactoside ( JP-A-08-134090).
  • kojic acid derivative in the present invention for example, kojic acid ester derivatives (Japanese Patent Publication Nos. 60-9722, 61-60801, JP 2000-344760, JP 2003-155 283), Kojic acid Ether derivatives (JP-A-3-14508), kojic acid derivatives substituted at the 2-position with mono- or dihydroxybenzoic acid (JP-A-07-188206), and dimers of kojic acid (JP-A 05-310727) Etc.
  • Kojic acid derivative salts and glycosides are also included in the kojic acid derivative of the present invention.
  • kojic acid or its derivatives, their salts and glycosides are collectively referred to as kojic acids.
  • the medium used in the present invention may be any serum-containing medium, serum-free medium, medium not containing animal protein-derived medium, protein-free medium, etc., as long as it can be used for animal cell culture.
  • a serum-free medium and a protein-free medium are preferable.
  • basal media used for normal animal cell culture used in the method of the present invention include RPMI1640 medium [The Journal of the American Medical Association, 199, 519 (1967)], Eagle's MEM Medium [Science, 122, 501 (19 52)], Dulbecco's modified MEM (DMEM) medium [Virology, 8, 396 (1959)], 199 medium [Proceeding of the Society for the Biological Medicine, 73, 1 (1950 ], F12 medium (manufactured by LTI) [Proc. Natl. Acad. Sci. USA, 53, 288 (196 5)], Iskov modified Dulbecco medium (IMDM medium) [J.
  • EX—CELL TM 302 medium manufactured by JRH or their modified or mixed medium, preferably RPMI1640 medium, DMEM, F12 medium, I MDM and EX-CELL TM 302 medium .
  • the serum-containing medium a medium containing the above basal medium supplemented with an appropriate amount of animal serum such as horsetail, horse, fish, etc., usually around 5 to 10%, is used.
  • nutrient factors examples include glucose, amino acids, vitamins and the like.
  • 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-leucine, L-lysine, L-methionine, L Examples include phenylalanine, L-pulin, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-parin, which can be used alone or in combination.
  • vitamins include d-piotine, D-pantothenic acid, choline, folic acid, myo-inositol, niacinamide, pyridoxal, riboflavin, thiamine, cyanocobalamin, DL- ⁇ tocopherol, and one or two of them Used in combination.
  • physiologically active substances include insulin, transferrin, serum albumin, and serum fractions containing growth factors.
  • physiologically active substances produced by genetic recombination methods, hydrolysates, or animal-derived materials may be used instead of animal-derived materials such as serum albumin and serum fractions. Examples thereof include a medium supplemented with lipids not contained.
  • hydrolyzate examples include hydrolysates such as soybean, wheat, rice, peas, cottonseed or yeast extract.
  • lipids examples include cholesterol, linoleic acid, and linolenic acid.
  • protein-free medium examples include ADPF medium (Animal derived protein free medium; HyClone), CD-Hybridoma medium (Invitrogen), CD-CHO medium (Invitrogen), and the like.
  • a medium containing a high concentration of amino acids and vitamins such as a medium in which RPMI1640 medium, DMEM medium and F12 medium are mixed at a ratio of 1: 1: 1, DMEM medium And F 12 medium mixed at a ratio of 1: 1 , NO, and hybridoma SFM medium (manufactured by Invitrogen) are preferably used.
  • the concentration in the case where at least one substance selected from lignans, flavonoids, histone deacetylase inhibitors, terpenoids, and kojic acids is added to the medium is usually 10 to 3000.
  • ⁇ mol / Preferably 30-2500 ⁇ mol / More preferably 50-2200 ⁇ mo ⁇ / Especially preferably 550 ⁇ molZL, but the type of animal cells used for culture, the type of substance produced, lignans, flavonoids
  • histone deacetylase inhibitor, terpenoids, and kojic acid can also be selected as appropriate according to the timing of addition of the at least one substance or the kind of additive.
  • Lignans, flavonoids, histone deacetylase inhibitors, terpenoids, kojic acid power The substance selected may be a single substance, or multiple substances may be added to the medium simultaneously or at different times. These substances may be added after being mixed.
  • a substance selected from lignans, flavonoids, histone deacetylase inhibitors, terpenoids, and kojic acids can be added to a medium before seeding the cells. It may be added to the medium at an appropriate time during the culture. Further, when a plurality of substances are combined, butyric acid, retinoic acid, and coenzyme Q10, which are known to improve the productivity of substances in animal cell culture, may be used.
  • the animal cell used in the present invention is any animal cell having the ability to produce a substance, such as an animal cell belonging to any of mammals, birds, reptiles, amphibians, fish, insects, etc.
  • animal cells belonging to mammals are preferably used.
  • animal cells derived from primates such as humans or monkeys or animal cells derived from rodents such as mice, rats or hamsters are preferably used.
  • Animal cells belonging to mammals include myeloma cells, ovarian cells, kidney cells, blood cells, uterine cells, connective tissue cells, mammary cells or embryonic retinoblasts, or cells derived from these cells.
  • myeloma cells or ovary cells, or cells derived from these cells are preferred.
  • antibody-producing cells such as hypridoma are preferably used.
  • Examples of animal cells belonging to mammals include the human cell line HL-60 (ATCC C CL—240), HT—1080 (ATCC CCL—121), HeLa (ATCC CCL—2), 293 (ECACC 85120602), Namalwa (ATCC CRL—1432), Namalwa KJM—1 (Cytotechnology, 1, 151 (1988) , NM—F9 cells (DSM ACC2605, WOO 5Z17130) and PER. C6 cells (ECACC No.
  • animal cells belonging to insects include moth (SOodoOt era frueiperda) cell line Sf 9 (ATCC CRL—
  • animal cells used in the present invention primary monkey kidney cells, primary rabbit rabbit cells, primary chicken embryo cells, primary quail embryo cells, which are primary culture cells used for vaccine production. Cells or the like may be used.
  • Examples of myeloma cells or cells derived from myeloma cells include Sp2Z0—Agl4, NS0, Y3 Agl. 2. 3., YO, and YB2Z0.
  • Examples of cells derived from ovary cells or oocyte cells include CHO-Kl, CHOZdhfr-, or CHOZDG44.
  • Kidney cells include 293, VERO, COS-7, BHK21, or MDCK
  • blood cells include HL-60, Namalwa, Namalwa KJM-l, or NM-F9.
  • Examples of force connective tissue cells include HT-1080 and NIH3T3.
  • Examples of force mammary cells include C1271I.
  • Examples of force embryonic retinoblasts include PER. C6.
  • Animal cells used in the present invention include animal cells that produce a substance, and a mutation treatment. Cells that have produced substances, cells transformed with thread-recombinant vectors that contain genes involved in substance production, fusion cells of antibody-producing cells such as B cells and myeloma cells A certain high-pridoma is used. Animal cells obtained by subjecting the cells of the present invention to a mutation treatment that increases the expression level of the substance may also be used.
  • 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.
  • a recombinant vector containing a DNA involved in the production of the substance and a promoter is used as the animal used in the present invention.
  • examples thereof include cells obtained by introduction into cells.
  • 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 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 be produced by animal cells, for example, biocatalytic molecules such as peptides and ribozymes, keratin, collagen, elastin, resilin, and five mouthin.
  • biocatalytic molecules such as peptides and ribozymes, keratin, collagen, elastin, resilin, and five mouthin.
  • ⁇ Formation of the cell structure of '' such as pressure ulcer vaccine, polio vaccine, measles vaccine, rubella vaccine, mumps vaccine, rabies vaccine, varicella vaccine, ushi epidemic fever vaccine, Ibaraki disease vaccine and ushi infectious tracheitis vaccine Examples include vaccines and viruses such as adenovirus.
  • Peptides produced by the method of the present invention include peptides derived from eukaryotic cells, preferably peptides derived from mammalian cells.
  • the peptide may be a desired peptide itself, a peptide containing the peptide, a fusion peptide fused with another peptide, or a partial fragment thereof.
  • Specific examples of the peptide produced by the method of the present invention include glycoprotein or physiological activity. And a peptide having sex.
  • glycoproteins include antibodies, erythropoietin (EPO) [J. Biol. Chem., 252, 5558 (1977)], ⁇ ombopoietin (TPO) [Nature, 369 533 (1994)] tissue Plasminogen activator, prolokinase, thrombomodulin, antithrombin III, 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 morphogenetic factor, condylar granulocyte colony stimulating factor (G — CSF) ⁇ .
  • EPO erythropoietin
  • TPO ⁇ ombopoietin
  • Examples of antibodies include V, antibodies having such antigen binding properties, such as antibodies that bind to tumor-related antigens, antibodies that bind to antigens related to allergy or inflammation, and antigens related to cardiovascular diseases. Examples include antibodies that bind, antibodies that bind to antigens associated with autoimmune diseases, or viruses! /! That bind to antigens associated with bacterial infections.
  • the antibody class may be any, but the IgG class is preferred.
  • the antibody produced by the method of the present invention includes a fragment containing a part of the antibody.
  • Fab abbreviation of Fragment of antigen binding
  • Fab fragment of antigen binding
  • Fab fragment of antigen binding
  • F abbreviation of F (ab,)
  • Single-chain antibodies single chain Fv; hereinafter referred to as scFv
  • dsFv disulfide stabilized antibodies
  • fusion proteins containing the Fc region of the antibody include white matter.
  • Examples of antibodies include antibodies produced by gene recombination techniques, in addition to antibodies secreted by high-pridoma cells produced from antibody-producing cells such as spleen cells of immunized animals after immunization of the animal with antigens, Examples thereof include antibodies obtained by introducing an antibody expression vector into which an antibody gene has been inserted into a host cell. Specific examples of the antibody include an antibody produced by Hypridoma, a human chimerized antibody, a human rabbit antibody, a human antibody, and the like.
  • a human chimeric antibody is a non-human animal antibody heavy chain variable region (hereinafter referred to as heavy chain as H chain, variable region as V region as HV or VH) and antibody light chain variable region (hereinafter referred to as "H chain”).
  • the light chain is also referred to as LV or VL as the L chain) and the heavy chain constant region of human antibodies (hereinafter, the constant region is also referred to as CH as C region) and the light chain constant region of human antibodies (hereinafter also referred to as CL).
  • Any animal other than a human can be used as long as it can produce a hybridoma, such as a mouse, rat, nomstar, or rabbit.
  • 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 hlgG class, and further higGl, hIgG2, hIgG3, Any of the subclasses such as hIgG4 can be used.
  • hlg human immunoglobulin
  • the CL of the human chimeric antibody any of those belonging to hlg can be used, and those of ⁇ class or fly class can be used.
  • VH and VL of a non-human animal antibody amino acid ti ⁇ U of human type homology determining region (hereinafter referred to as CDR) is designated as VH of human antibody.
  • CDR human type homology determining region
  • Human-type CDR-grafted antibody is a cDNA encoding the V region in which the VH and VL CDR sequences of non-human animal antibodies are grafted to the VH and VL CDR sequences of any human antibody. And constructing a human CDR-grafted antibody expression vector by inserting it into a host cell expression vector having genes encoding human antibody CH and human antibody CL, and introducing the expression vector into the host cell. To express and produce human CDR-grafted antibodies.
  • any CH can be used as long as it belongs to hlg, but the hlgG class is preferable, and hlgG 1, hIgG2, hIgG3, and hlg G4 belonging to the hlgG class are preferred. Either can be used.
  • the CL of the human CDR-grafted antibody any ⁇ class or ⁇ class antibody belonging to hlg can be used.
  • antibodies produced by the method of the present invention include anti-GD2 antibodies [Anticancer Res., 13, 331 (1993)], anti-GD3 antibodies [Cancer Immunol] Immunother., 36, 260 (1993)], anti-GM2 antibody [Cancer Res., 54, 1511 (1994)], anti-HER2 antibody [Proc. Natl. Acad. Sci. USA, 89, 4 285 (1992)] , Anti-CD52 antibody [Nature, 332, 323 (1988)], anti-MAGE antibody (British J. Cancer, 83, 493 (2000)), anti-HMl.
  • antibodies that bind to antigens associated with allergies or inflammation include anti-ins Turleukin 6 antibody [Immunol. Rev., 127, 5 (1992)], anti-interleukin 6 receptor antibody [Molecular Immunol., 31, 371 (1994)], anti-interleukin 5 antibody [I mmunol. Rev., 127 , 5 (1992)], anti-interleukin 5 receptor antibody, anti-interleukin 4 antibody [Cytokine, 3, 562 (1991)], anti-interleukin 4 receptor antibody ⁇ . Im munol.
  • antibodies that bind to an antigen associated with cardiovascular disease include anti-GpIIbZlIIa antibody Ci. Immunol., 152, 2968 (1994)], anti-platelet-derived growth factor antibody [Science, 253.1129 ( 1991)], antiplatelet-derived growth factor receptor antibody Q [. Biol. Chem., 272, 17400 (1997)] or anti-blood coagulation factor antibody [Circulation, 101, 1158 (2000)].
  • antibodies that bind to antigens associated with autoimmune diseases include anti-self DNA antibodies [Immunol. Letters, 72, 61 (2000)], anti-CDlla antibodies, anti-ICAM3 antibodies, anti-CD80.
  • Antibody, anti-CD2 antibody, anti-CD3 antibody, anti-CD4 antibody, anti-integrin ⁇ 4 j87 antibody, anti-CD40L antibody, anti-IL 2 receptor antibody [Immunology Today, 21, 403 (2000)].
  • antibodies that bind to an antigen associated with a virus or bacterial infection include anti-gpi 20 antibody [Structure, 8, 385 (2000)], anti-CD4 antibody ⁇ . Rheumatology, 25, 2 065 (1998). )], Anti-CCR4 antibody, anti-verotoxin antibody Q [. Clin. Microbiol., 37, 396 (1999)].
  • the peptide having physiological activity is not particularly limited, and examples thereof include peptides that maintain the activity of the glycoprotein among the partial fragments of the glycoprotein.
  • a peptide or enzyme structure that regulates the enzyme activity Peptides that retain the structure are also included.
  • Examples of peptides that regulate the activity of the enzyme include peptides that function as glycoprotein agonists or antagonists.
  • Examples of agonists include peptides having the activity of enhancing glycoprotein activity, and specifically include somatostatin derivatives, somatrobin, atrial natriuretic peptide, glucagon, insulin, insulin-like growth factor, gonadotropin-releasing hormone. Lemon.
  • the antagonist include peptides having an activity of suppressing the activity of glycoprotein, and specifically, pegpisomanto.
  • the animal cell used in producing the peptide by the method of the present invention contains a gene that codes the peptide to be produced, although the animal cell may be used as long as the peptide can be produced.
  • a transformed cell into which a vector containing a gene encoding a peptide that is preferably used is introduced can be obtained by using, for example, a thread-recombinant vector containing a DNA encoding a peptide and a promoter. , Obtained by introducing into the host cell.
  • the animal cells described above are used as host cells.
  • Examples of the vector used for preparing a vector containing a gene encoding a peptide include pcDNAI, pcDM8 (manufactured by Funakoshi), pAGE107 [Japanese Patent Laid-Open No. 3-22979, Cytotechnology, 3, 133 ( 1990)], pAS3-3 (JP-A-2-227075), pcDM8 [Nature, 329. 840 (1987)], pcDNAlZAmp (Invitrogen), pREP4 (Invitrogen), pAGE103 Q [. Biochem., 101 , 1307 (1987)], and ⁇ AGE210.
  • Any promoter can be used as long as it functions in animal cells used in the present invention.
  • CMV cytomegalovirus
  • IE immediate early gene promoter
  • early SV40 Examples include promoters, retrowinoles promoters, meta-mouthone promoters, heat shock promoters, SRa promoters, and the like.
  • an enhancer of the IE gene of human CMV may be used together with a promoter.
  • DNA is introduced into the cell.
  • Any method can be used, for example, the electopore position method [Cytot echnology, 3, 133 (1990)], the calcium phosphate method (JP-A-2-227075), the lipofusion method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987), Virology, 52, 4 56 (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), transformant cells producing anti-CCR4 humanized antibodies KM8759 (FERM BP—8129), 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 998399 (FERM BP—5648) And KM9399 (FERM BP—5647), transformed cells producing anti-GM2 human CDR-grafted antibody KM8966 (FERM BP—5105), KM8967 (FERM BP—5106), K M8969 (FERM BP—5527), KM
  • any culture method such as batch culture, repeat batch culture, food batch culture, and perfusion culture can be used as long as it can produce a desired substance.
  • food batch culture or perfusion culture is preferably used.
  • the Fuedbachi culture is a culture method in which physiologically active substances, nutrient factors, etc. are additionally supplied in small amounts continuously or intermittently.
  • the Fuedbatch culture can prevent a decrease in the cell density of the cultured cells due to accumulation of waste products in a culture solution that increases the metabolic efficiency of the cells.
  • the desired substance in the collected culture medium has a higher concentration than that obtained in notch culture, the substance can be easily separated and purified, and compared to notch culture, The production amount of the substance can be increased.
  • rydanans, flavonoids, histone deacetylase inhibitors, terpenoids, koji It is easier to control the concentration of at least one substance whose acid power is selected than batch culture.
  • Perfusion culture is efficiently separated by a device that separates the culture solution and cells, the concentrated cells are returned to the original culture tank, and the reduced amount of fresh medium is newly supplied to the culture tank.
  • the culture environment in the culture tank is always kept good, and the osmotic pressure in the tank can be controlled by supplying a fresh medium, so that lignans, flavonoids, and histones in the medium are removed.
  • Acetylenic enzyme inhibitor, terpenoids, kojic acid power is a preferred method for controlling the concentration of at least one selected substance
  • the culture method used in the present invention may be any method as long as it is suitable for the animal cells to be used, but usually 3 to 20 days under conditions of pH 6 to 8, 30 to 40 ° C, etc. In perfusion culture, carry out for 3 to 60 days.
  • antibiotics such as streptomycin and benicillin may be added to the medium as needed during culture.
  • the dissolved oxygen concentration control, pH control, temperature control, stirring, etc. can be carried out according to the method used for normal animal cell culture.
  • the desired substance can be produced by culturing animal cells having the above, producing and accumulating the desired substance in the culture, and collecting the desired substance from the culture.
  • the production method of the present invention includes a direct expression method in which the peptide is produced in the host cell, a method in which the peptide is secreted and produced outside the host cell (Molecular 'Cloning 2nd Edition). Etc.
  • Peptides can be prepared by the method of Paulson et al., I. Biol. Chem., 264, 17619 (1989)], the method of Law et al. [Proc. Natl. Acad. Sci. USA, 86, 8227 (1989), Genes Develop. , 4, 1288 (1990)], or by applying the method described in JP-A-5-336963, WO94Z23021 etc., it can be actively secreted outside the host cell.
  • a signal peptide is bound to the N-terminus of the desired peptide using a gene recombination technique. By expressing in the form, the peptide can be actively secreted outside the host cell.
  • the 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 cells are collected by centrifugation after culturing, suspended in an aqueous buffer, and then subjected to an ultrasonic crusher, Crush the cells with a French press, a Menton Gaurin homogenizer, or Dynomill to obtain a cell-free extract.
  • an ordinary enzyme isolation and purification method that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent,
  • Anion-exchange chromatography using resin such as Jetylaminoethyl (DEAE) -Sepharose, DIAION HPA-75 (Mitsubishi Kasei), Cation using resin such as S-Sepharose FF (Falmasia)
  • An exchange chromatography method a hydrophobic chromatography method using a resin such as butyl sepharose, ferrule sepharose, a gel filtration method using a molecular sieve, a affinity chromatography method using protein A, a chromatofocusing method,
  • an electrophoretic method such as Tatsuta Electrophoresis alone or in combination, a crudely purified sample or a purified sample Can be obtained.
  • the peptide produced by the method of the present invention is secreted extracellularly, the peptide can be recovered in the culture supernatant. That is, a culture supernatant is obtained by treating the culture by a technique such as centrifugation as described above, and a crudely purified sample is obtained from the culture supernatant by using the same isolation and purification method as described above. Alternatively, a purified sample can be obtained.
  • the productivity of the peptide can be improved by increasing the specific product rate (SPR) of the peptide.
  • SPR specific product rate
  • the SPR of a substance is calculated by the amount of substance produced per substance producing cell. Specifically, the amount of substance produced throughout the culture period is the amount of material that survived during the culture period. Calculated by dividing by the number of quality cells produced.
  • the productivity of substances per animal cell can be improved.
  • the medium for expansion until the main culture is EX-CELL TM 302 medium (manufactured by JRH), Met hotrexate (hereinafter referred to as MTX) 500 nmol / L (manufactured by Sigma-Aldrich), L glutamine (Wako Pure Chemicals) 1.
  • a medium supplemented with 75 g ZL was used. Place approximately 10-30% of the above medium in a 125 mL, 250 mL or 1 OOOmL Erlenmeyer flask (manufactured by Corning Co., Ltd.) to obtain 3 X 10 5 cells / mL. 503 LCA- 500D strain cells
  • the suspension was seeded. Incubate at 35 ° C for 4 days and pass several times until the number of cells required for seeding of the main culture is obtained.
  • the medium for the main culture includes a medium modified from EX-CELL TM 302 medium (manufactured by JRH; hereinafter referred to as modified EX—CELL TM 302 medium), MTX (manufactured by Sigma) 500 nmolZL, L— Glutamine (Wako Pure Chemical Industries, Ltd.) 1.
  • a medium supplemented with 75 gZL was used.
  • the feed medium contained amino acid (L-alanine 0.14 gZL, L-arginine monohydrochloride 0.47 gZL, L-asparagine monohydrate 0.16 gZL, L-aspartic acid 0.17 gZL, L-cystine dihydrochloride 0.51 g / L.
  • A: L-glutamic acid 0.42 g / L, L glutamine 7.3 g / L, glycine 0.17 g / L, L histidine monohydrochloride dihydrate 0.2 gZL, L-isoleucine 0.59 g ZL, L bite Ishin 0.5 9gZL, L-Lysine Monohydrochloride 0.82gZL, L Methionine 0.17gZL, L-Ferulalanine 0.37gZL, L Proline 0.22g / L-Serine 0.24g / L, L-Threo- 0.5 3gZL, L Tryptophan 0.09g / L, L-tyrosine disodium dihydrate 0.58g / L , L-parin 0.53 g / L), vitamins (d-piotin 0.073 mg ZL, D-pantothenic acid power Norecicum 0.02 g / L, salt ⁇ f gin 0.022 g / L, foli
  • VALPRO After adding VALPRO to the medium, it was cultured for 9 days by blowing 35 ° C, 100rpm, 5% CO.
  • control culture in a medium
  • the culture broth was collected on days 3, 5, 7 and 9 from the beginning of the culture, and the viable cell density (cell ZmL) and the concentration of the produced antibody (mgZL) were measured.
  • the viable cell density is a dye exclusion method using 0.4% trypan blue solution (manufactured by Invitrogen), and the concentration of the produced antibody (hereinafter also referred to as substance concentration) is determined by HPLC (manufactured by Shimadzu Corporation). Each was measured.
  • the cumulative cell density was shown as the sum of the product of the viable cell density and the elapsed time.
  • the viable cell density was measured on the third, fifth, seventh, and ninth days from the start of the culture, and the cumulative cell density was calculated from the following formula 1 using the measured viable cell density.
  • Cumulative cell density (cell ZmLZ day) (seeding cell density + measured cell density) X elapsed time ⁇ 2
  • the specific production rate was calculated from the following formula 2.
  • pgZ cell Z day substance concentration (mgZL) ⁇ cumulative cell density (cell ZmLZ ⁇ )
  • the viability showed a good value of 80% or more until the ninth day of culture, and the viability was 82% in the control culture on the 14th day, whereas it was 94% in the culture supplemented with valproic acid. Atsuta. The maximum cell density reached was 4.5 x 10 6 cells on day 7 in the control culture, whereas 1.8 x 10 6 on day 7 in the culture supplemented with valproic acid. Cells / day.
  • the medium for expansion until the main culture is EX-CELL TM 302 medium (manufactured by JRH), Met hotrexate (hereinafter referred to as MTX) 500 nmol / L (manufactured by Sigma Aldrich), L-glutamine (Jun Wako) 1.
  • MTX Met hotrexate
  • L-glutamine Jun Wako 1.
  • a medium supplemented with 75 g ZL was used. Place approximately 10-30% of the above medium in a 125 mL, 250 mL, or 1 OOOmL Erlenmeyer flask (manufactured by Corning), and a cell suspension of Ms704-CD20 strain to 3 X 10 5 cells ZmL Sowing.
  • 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.
  • the basic medium of the main culture includes a modified medium of EX-CELL TM 302 medium (manufactured by JRH; hereinafter referred to as modified EX-CELL TM 302 medium), MTX (manufactured by Sigma) 500 nmolZL, L —Glutamine (Wako Pure Chemical Industries, Ltd.) 1. A medium supplemented with 75 g / L was used.
  • the feed medium contained ⁇ amino acids (L-alanine 0.14 g / L-arginine monohydrochloride 0.47 g / L-asparagine monohydrate 0.16 gZL, L-aspartic acid 0.17 gZL, L-cystine dihydrochloride 0.5 lg / L-glutamic acid 0.42 g / L, L-glutamine 7.3 g / L, glycine 0.17 g / L, L-histidine monohydrochloride dihydrate 0.24 gZL, L-isoleucine 0.59 gZL, L-leu Shin 0.59gZL, L Dizine Monohydrochloride 0.82gZL, L-methionine 0.17gZL, L-Hue-Lulan 0.37gZL, L Proline 0.22gZL, L Serine 0.24g / L, L-Threonine 0.5gZL, L tryptophan 0.09g / L, L-ty
  • the antibody concentration was increased to 104 to 145% of the control.
  • the specific production rate of the antibody showed a productivity of 127 to 239% in all substances shown in the table as compared with the control.
  • the following fed-batch culture was performed using Ms705-pKAN- ⁇ strain (FERM-BP8472) having the ability to produce ATIII (antithrombin III).
  • EX-CELL TM 302 medium (manufactured by JRH) was used as the medium for expansion until the main culture.
  • Methotrexate hereinafter referred to as MTX
  • L-glutamine Wang Chemical Industries
  • 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.
  • modified EX-CELL TM 302 medium A medium obtained by modifying EX-CELL TM 302 medium (manufactured by JRH; hereinafter referred to as modified EX-CELL TM 302 medium) was used as the basic medium for the main culture.
  • MTX Sigma 500 nmolZL and L-glutamine (Wako Pure Chemical Industries) 1.75 g / L were added to the modified EX-CELL T M 302 medium.
  • the feed medium contained amino acids (L-alanine 0.14 g / L, L-arginine monohydrochloride 0.47 gZL, L-parasparagin monohydrate 0.16 gZL, L-parasine acid 0.17 gZL, L-cystine dihydrochloride 0.
  • control culture in medium
  • camphor concentrate dissolved in a ratio of 334.9 mg of camphor in 1 mL of DMSO (manufactured by Sigma-Aldrich) was used.
  • the culture broth was collected on days 3, 5, 7, 9, 11, and 14 from the beginning of the culture, and the viable cell density (cell ZmL) and the concentration of sputum produced (mgZL) were measured.
  • Live cells Density is a dye exclusion method using 0.4% trypan blue solution (manufactured by Invitrogen).
  • the concentration of soot produced (hereinafter also referred to as substance concentration) is determined by HPLC (manufactured by Shimadzu Corporation). Each was measured.
  • the cumulative cell density was expressed as the sum of the product of the viable cell density and the elapsed time.
  • the viable cell density was measured on the third, fifth, seventh, ninth, eleventh, and fourteenth days from the start of the culture, and the cumulative cell density was calculated using the measured viable cell density using the following formula 3 Calculated by
  • Cumulative cell density (cell ZmLZ day) (culture day 0 cell density + culture day 3 cell density) / 2 (3—0) day + (culture day 3 cell density + culture day 5 cell density) 72 ( 5-3) day + ' ⁇ ⁇ + (cell density on day 11 of culture + cell density on day of culture 14) Z2 X (14–11) day
  • the specific production rate was accumulated in the culture supernatant at the end of the culture
  • the substance concentration (mgZL) was calculated by dividing by the cumulative cell density (cell ZmLZ day).
  • the viability showed a good value of 90% or more until the 10th day of culture, and the viability was 79% in the control culture on the 14th day, whereas 91% in the culture supplemented with camphor. It was hot.
  • the maximum cell density reached was 7.4 x 10 6 cells Z on day 11 in the control culture, whereas on day 14 in culture supplemented with camphor at a concentration of 0.55 mmol / L. 7. 4 ⁇ 10 6 cells Z days.
  • Fig. 3 shows the concentration (mg / L) of sputum produced relative to the cumulative cell density (cells / mL x day) on day 11 and day 14 after the start of culture.
  • the cumulative cell density was 5.8 ⁇ 10 6 cells ZmLZ day in 14 days in the control culture, and the concentration of sputum produced was 985 mgZL, so the specific production rate was 170 pgZ cell Z days.
  • the cumulative viable cell density was 4.3 X 10 6 cells ZmLZ day in 14 days, and the concentration of sputum produced was 1078 mgZL From that The specific production rate was 251 pgZ cells Z days.
  • the Ms704—CD20 strain producing the anti-CD20 antibody (FERM BP-10092) and the 709 LCA-500G strain producing the anti-CCR4 humanized antibody were used as described in Example 3.
  • the fed-batch culture was performed in the same manner as the method. However, camphor was added at a concentration of 0.55 mmol / L.
  • the 709 LCA-500G strain was obtained in the same manner as the 503LCA-500D strain described in Example 3 of WO03Z046174.
  • the cumulative cell density on the 14th day from the start of the culture was 5.8 ⁇ 10 6 cells / mLZ, Since the antibody concentration was 985 mgZL, the specific production rate was 170 pgZ cells / day, whereas in the culture supplemented with camphor, the cumulative viable cell density on the 14th day from the start of culture was 4.3.
  • X 10 6 cells were ZmLZ days, and the concentration of antibody produced was 1078 mgZL, and the specific production rate was 251 pgZ cells Z days.
  • Fig. 4 shows the specific production rate of the produced anti-CD20 antibody in the culture at the start of the culture on day 14 with or without camphor.
  • the cumulative cell density was 4.7 X 10 6 cells / mL Z day on the 14th day from the start of the culture. Since the antibody concentration was 590 mgZL, the specific production rate was 126 pgZ cells / day, whereas in the culture supplemented with camphor, the cumulative viable cell density was 2.OX 10 on the 14th day from the start of culture. Since the 6- cell ZmLZ day and the antibody concentration was 425 mgZL, the specific production rate was 213 pgZ-cell Z-day. On the 14th day after the start of culture, the anti-CCR4 humanized antibody produced in the culture with or without camphor was added. The specific production rate is shown in Fig. 5.
  • Food batch culture was performed in the same manner as in Example 4 using Ms704-CD20 strain (FERM BP-10092) that produces anti-CD20 antibody.
  • Fig. 6 shows the number of viable cells in culture for each period when camphor was added.
  • the viability of the cells showed a good value of 90% or more until the 10th day of culture, which was 79% in the control culture on the 14th day, whereas the culture start power was 91% in the culture with the addition of camphor. %Met.
  • the maximum density reached in the control culture was 7.4 x 10 6 cells on day 11 from the start of culture, whereas in the culture with camphor added, it was 7.4 x 10 6 on day 14 from the start of culture. Since the number of days to reach the maximum density was extended by adding camphor at the initial stage of culture, the cell growth was suppressed by the addition of camphor. It became power.
  • Fig. 7 shows the specific production rate of the substance at each time when camphor was added. As shown in Fig. 7, the specific antibody production rate was highest when camphor was added at the same time as the start of culture, and when camphor was added within 5 days from the start of culture, The productivity improvement effect was recognized. On the other hand, when camphor was added after 7 days from the start of culture, no significant improvement in antibody productivity was observed.
  • camphor when camphor is added to a culture medium and a substance such as an antibody is cultured for production, the camphor is added at the initial stage of the culture, that is, when the cells are actively proliferating.
  • the addition of camphor at the initial stage of the culture suppressed cell growth. Therefore, The timing for adding camphor to the medium needs to be set in consideration of the growth of cells used for production and the specific production rate.
  • Serum-free fed-batch culture of anti-CCR4 human chimeric antibody using 709 LCA-500G strain The following fed-batch culture was performed using 709 LCA-500G strain having the ability to produce anti-CCR4 human chimeric antibody.
  • the 709 LCA-500G strain was obtained by the same method as the 503 LCA-500D strain (FERM BP-8239) described in Example 3 of WO03Z046174.
  • EX-CELL TM 302 medium (manufactured by JRH) was used as the medium for expansion until the main culture.
  • Methotrexate hereinafter referred to as MTX
  • L-glutamine Wang Chemical Industries
  • 1 Place approximately 10-30% of the above medium in a 25 mL, 250 mL or lOOOmL Erlenmeyer flask (manufactured by Corning Co., Ltd.), and 709 LCA-500G cell suspension to give 3 X 10 5 cells ZmL
  • the suspension was seeded.
  • 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.
  • modified EX-CELL TM 302 medium A medium obtained by modifying EX-CELL TM 302 medium (manufactured by JRH; hereinafter referred to as modified EX-CELL TM 302 medium) was used as the basic medium for the main culture.
  • MTX Sigma 500 nmolZL and L-glutamine (Wako Pure Chemical Industries) 1.75 g / L were added to the modified EX-CELL T M 302 medium.
  • the feed medium contained amino acids (L-alanine 0.14 g / L, L-arginine monohydrochloride 0.47 gZL, L-parasparagin monohydrate 0.16 gZL, L-parasine acid 0.17 gZL, L-cystine dihydrochloride 0.
  • the culture medium was collected on days 3, 5, 7, 9, 11, 13, and 15 from the beginning of the culture, and the density of live cells (cell ZmL) and the concentration of produced antibody (mgZL) were measured.
  • the density of live cells is a dye exclusion method using 0.4% trypan blue solution (manufactured by Invitrogen).
  • the concentration of the antibody produced (hereinafter also referred to as substance concentration) is HPLC (manufactured by Shimadzu Corporation). Respectively.
  • the cumulative cell density was expressed as the sum of the product of the viable cell density and the elapsed time.
  • the viable cell density was measured on the third, fifth, seventh, ninth, eleventh, thirteenth, and fifteenth days from the start of the culture, and the cumulative cell density was measured using the measured live cell density as follows. Calculated according to Equation 4.
  • Cumulative cell density (cell ZmLZ day) (culture day 0 cell density + culture day 3 cell density) / 2 (3-0) day + (culture day 3 cell density + culture day 5 cell density) 72 (5-3) day + ' ⁇ ⁇ + (culture day 13 cell density + culture day 15 cell density)
  • the Z2X (15-13) day specific production rate was calculated by dividing the concentration of the substance accumulated in the culture supernatant (mgZL) at the end of the culture by the cumulative cell density (cell ZmLZ day).
  • Figure 8 shows the time course of the density of viable cells when kojic acid was added at a concentration of ImmolZL.
  • the maximum cell density reached was 2.9 X 10 6 cells / day on day 11 in the control culture, whereas 2.0 to 3.4 X on day 9 in the culture supplemented with kojic acid. 10 6 cells Z days.
  • the cell viability in the culture showed a good value of 80% or more from the start of the culture to the 9th day of culture, and on the 15th day it was 53% in the control culture, whereas kojic acid was added. In cultivated cultures, it was 29-57%.
  • the calculated specific production rate of the antibody is shown in Fig. 9 for each kojic acid addition concentration and each addition time.
  • the specific production rate of the antibody increased depending on the concentration of the added kojic acid, and the antibody productivity was significantly improved compared to the control. there were.
  • the effect of improving antibody productivity was not significant, and was not proportional to the concentration of added kojic acid.
  • the specific antibody production rate is as follows: 1. When kojic acid was added on the 3rd day from the start of culture at a concentration of OmmolZL, the effect of improving the antibody productivity by the addition of kojic acid was confirmed. It was. However, in this culture condition where the antibody productivity improvement effect was maximized, the viable cell density during the culture period decreased compared to the other culture conditions as shown in FIG. . In addition, the cell viability on the 15th day after the start of culture was the lowest at 27%.
  • Table 1 shows the concentrations of substances accumulated in the culture supernatant at the end of the 15th day of culture (hereinafter referred to as cumulative production antibody concentration). In addition, cumulative production for control culture The ratio of antibody amount is also shown.
  • productivity of a substance to be produced can be improved in a method for culturing animal cells and producing the cellular force substance.

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Abstract

Disclosed is a method for production of a substance, which is characterized by cultivating an animal cell in a culture medium supplemented with at least one substance selected from a lignan, a flavonoid, a histone deacetylase inhibitor, a terpenoid and kojic acid or a derivative thereof. Also disclosed is a method for improvement of the productivity of a substance, which is characterized by cultivating an animal cell in a culture medium supplemented with at least one substance selected from a lignan, a flavonoid, a histone deacetylase inhibitor, a terpenoid and kojic acid or a derivative thereof.

Description

明 細 書  Specification
物質の製造方法  Method for producing substance
技術分野  Technical field
[0001] 本発明は、リグナン類、フラボノイド類、ヒストン脱ァセチルイ匕酵素阻害剤、テルぺノ イド類、コウジ酸またはその誘導体力 選ばれる物質を添加した培地中で動物細胞 を培養することを特徴とする物質の製造方法、およびリグナン類、フラボノイド類、ヒス トン脱ァセチルイ匕酵素阻害剤、テルぺノイド類、コウジ酸またはその誘導体カゝら選ば れる物質力も選ばれる物質を添加した培地を用いることを特徴とする、物質の細胞あ たりの生産性を向上させる方法に関する。  [0001] The present invention is characterized by culturing animal cells in a medium to which a substance selected from lignans, flavonoids, histone deacetylase inhibitors, terpenoids, kojic acid or derivatives thereof is added. And a medium supplemented with a substance selected for the substance strength selected from lignans, flavonoids, histone deacetylase inhibitors, terpenoids, kojic acid or derivatives thereof. The present invention relates to a method for improving the productivity of a substance per cell.
背景技術  Background art
[0002] ペプチドは、さまざまな医薬品用途を示し、特に抗体などの免疫機能を有するぺプ チドは、腎移植の拒絶反応の低減化、 RSV小児感染に対する抗ウィルス剤、乳がん に対する抗ガン剤などの医薬品に応用されている。そのなかでも、抗体は医薬として その重要性が今後益々高くなると予想されている。  [0002] Peptides have various pharmaceutical uses. In particular, peptides with immune functions such as antibodies are used to reduce rejection of renal transplantation, antiviral agents against RSV childhood infection, anticancer agents against breast cancer, etc. Applied to pharmaceuticals. Among them, antibodies are expected to become increasingly important as pharmaceuticals.
抗体などの糖蛋白質をコードする遺伝子を用 ヽて遺伝子組換え糖蛋白質を製造 するには、糖蛋白質をコードする遺伝子を導入した組換え細胞を培養し、培養物中 に生産される遺伝子組換え糖蛋白質を採取することにより行われるが、遺伝子組換 え糖蛋白質は動物細胞でしか完全な成熟体として生産されることはないため、遺伝 子組換え糖蛋白質の多くは動物細胞を用いて製造されて 、る。  In order to produce a recombinant glycoprotein using a gene encoding a glycoprotein, such as an antibody, a recombinant cell into which the gene encoding the glycoprotein has been introduced is cultured, and the recombinant protein produced in the culture is produced. Although it is performed by collecting glycoproteins, genetically engineered glycoproteins are produced only in animal cells as fully mature forms, so many genetically modified glycoproteins are produced using animal cells. It has been.
[0003] 糖蛋白質のなかでも抗体は、特に近年、種々の医薬品として認可され、さらには多 くの候補抗体が種々の開発ステージに到達しており（非特許文献 1)、今後、動物細 胞での抗体などの有用物質の生産が益々盛んになることが予測されているが、動物 細胞による有用物質の生産性はまだ充分に高くないことが問題点としてあげられてい る。  [0003] Among glycoproteins, antibodies have recently been approved as various pharmaceuticals, and many candidate antibodies have reached various development stages (Non-patent Document 1). It is predicted that the production of useful substances such as antibodies will increase more and more, but the problem is that the productivity of useful substances by animal cells is not yet high enough.
これらの問題点を解決し、供給量を確保する方法としては大型の培養槽を用いる方 法 (非特許文献 2)が挙げられるが、このような大型培養槽での動物細胞の培養は、 細胞への酸素の供給不足または培地中の溶存炭酸ガスの増大などの問題がある。 [0004] また、培養中の動物細胞が産出する乳酸やアンモニアに代表される副産物は動物 細胞の増殖に阻害を示すことから、アンモニアの発生源であるグルタミン不含培地で 生育可能なグルタミンシンセターゼ遺伝子を導入した細胞 (特許文献 1)が、医薬品 の製造に用いられており、このような工夫により主に培養延長などで効果が得られて いる。ペプチドの生産に多く用いられる CHO細胞またはミエローマ細胞などの哺乳 類動物細胞を用いて、細胞あたりのペプチドなどの物質の生産性を向上させるため、 発現ベクター系、細胞選択方法、培養方法、精製方法などの検討が行われている（ 非特許文献 3)。例えば、哺乳類動物細胞用培地の浸透圧は約 260〜320 mOsm /kgが一般的とされる力 浸透圧が 450〜600 mOsmZkgなど高い時に、物質の 細胞あたりの生産性が向上することが知られている（特許文献 3、特許文献 4)。 As a method for solving these problems and securing the supply amount, there is a method using a large culture tank (Non-patent Document 2). Animal cell culture in such a large culture tank is a cell culture method. There is a problem such as insufficient supply of oxygen to the water or an increase in dissolved carbon dioxide in the medium. [0004] In addition, since by-products such as lactic acid and ammonia produced by animal cells in culture show inhibition of animal cell growth, glutamine synthetase that can grow on a glutamine-free medium that is a source of ammonia Cells into which a gene has been introduced (Patent Document 1) are used for the production of pharmaceuticals, and such a device has been effective mainly by extending culture. In order to improve the productivity of substances such as peptides per cell using mammalian cells such as CHO cells or myeloma cells often used for peptide production, expression vector systems, cell selection methods, culture methods, purification methods (Non-Patent Document 3). For example, the osmotic pressure of mammalian cell culture medium is generally about 260-320 mOsm / kg. It is known that the productivity per substance of cells improves when the osmotic pressure is high, such as 450-600 mOsmZkg. (Patent Literature 3, Patent Literature 4).
[0005] CHO細胞またはミエローマ細胞などの哺乳類動物細胞を用いてペプチドなどの物 質の生産性を向上させる方法としては、種々の生産性を向上させる物質を培地中に 添加する方法が知られている。具体的には、酪酸 (特許文献 5)、レチノイン酸 (特許 文献 6)、コェンザィム Q10 (特許文献 7)などの物質を添加して培養する方法などが 知られている。  [0005] As a method for improving the productivity of a substance such as a peptide using mammalian cells such as CHO cells or myeloma cells, a method of adding various substances that improve productivity to the medium is known. Yes. Specifically, a method of culturing by adding substances such as butyric acid (Patent Document 5), retinoic acid (Patent Document 6), and Coenzyme Q10 (Patent Document 7) is known.
[0006] ノ レプロ酸およびその誘導体はヒストン脱ァセチルイ匕酵素の阻害剤であり、抗てん 力ん薬として知られている (特許文献 8)。セサモールは糖'脂質代謝改善剤として知 られている（特許文献 9)。ルテオリン、ァカセチンなどのフラボン類は感染症の治療 を目的とした化合物として知られている（特許文献 10)。ダイゼインなどのイソフラボン は女性ホルモン産生促進剤として皮膚外用剤に配合されることが知られて！/ヽる（特許 文献 11)。オンジサポニン、グリチルレチン酸もそれぞれ皮膚外用剤に添加されるこ とが知られている（特許文献 12、特許文献 13)。しかし、いずれの物質についても、 培地に添加して動物細胞を培養した場合、物質の細胞あたりの生産性へどのような 影響があるかは知られて ヽな 、。  [0006] Noreproic acid and its derivatives are inhibitors of histone deacetylase, and are known as anti-epileptics (Patent Document 8). Sesamol is known as a sugar-lipid metabolism improving agent (Patent Document 9). Flavones such as luteolin and acacetine are known as compounds aimed at treating infectious diseases (Patent Document 10). Isoflavones such as daidzein are known to be incorporated into topical skin preparations as female hormone production promoters! / Speak (Patent Document 11). It is known that ondisaponin and glycyrrhetinic acid are also added to the external preparation for skin (Patent Documents 12 and 13). However, it is well known that any substance has an effect on the per-cell productivity of animal cells when added to the medium and cultured in animal cells.
[0007] テルぺノイド類のうち、カンファは誘導刺激薬や角膜障害を予防するための局所適 用製剤として用いられることが知られている（非特許文献 4、特許文献 14)。カンファ は培地に添加すると、増殖阻害 (非特許文献 5)、増殖促進 (非特許文献 6)といった 、相反する細胞の増殖への影響があることが知られている。し力しながら、カンファを 培地に添加して動物細胞を培養した場合、物質の細胞あたりの生産性に対して、ど のような影響があるかは知られて ヽな 、。 [0007] Among terpenoids, camphor is known to be used as a topical preparation for preventing induced stimulants and corneal disorders (Non-patent Document 4, Patent Document 14). It is known that when camphor is added to a medium, it has an influence on the proliferation of conflicting cells such as growth inhibition (Non-patent document 5) and growth promotion (Non-patent document 6). While working hard, It is well known how animal cells are cultivated by adding them to the medium, which has an effect on the productivity of the substance per cell.
コウジ酸またはその誘導体は美白剤として知られている（特許文献 15)。また、化学 合成培地に添カ卩したトランスフェリンへのキレート効果を検討するための、コウジ酸の 培地への添加が知られている（特許文献 16)。この場合、コウジ酸の添カ卩により、 CH O細胞の増殖が阻害されたことが報告されているが、物質の細胞あたりの生産性に 対するコウジ酸の影響についての記載はなぐコウジ酸またはその誘導体を培地に 添加して動物細胞を培養した場合、物質の細胞あたりの生産性へどのような影響が あるかは知られていない。  Kojic acid or its derivatives are known as whitening agents (Patent Document 15). In addition, the addition of kojic acid to the medium for the purpose of examining the chelating effect on transferrin added to the chemically synthesized medium is known (Patent Document 16). In this case, the addition of kojic acid has been reported to inhibit the growth of CHO cells, but there is no description of the effect of kojic acid on the per-cell productivity of the substance. When animal cells are cultured with the derivative added to the medium, it is not known what effect the substance has on the productivity per cell.
特許文献 1 :US5747308 Patent Document 1: US5747308
特許文献 2:WO01/29246 Patent Document 2: WO01 / 29246
特許文献 3： W096Z39488 Patent Document 3: W096Z39488
特許文献 4： US4724206 Patent Document 4: US4724206
特許文献 5：特開平 8 -9968 Patent Document 5: JP-A-8-9968
特許文献 6:US5155136 Patent Document 6: US5155136
特許文献 7： WO03/046174 Patent Document 7: WO03 / 046174
特許文献 8： WO02/007722 Patent Document 8: WO02 / 007722
特許文献 9：特開平 11 246427 Patent Document 9: JP-A-11 246427
特許文献 10:WO0lZ003681 Patent Document 10: WO0lZ003681
特許文献 11:特開 2004— 67590 Patent Document 11: Japanese Unexamined Patent Application Publication No. 2004-67590
特許文献 12:特開平 8— 133948 Patent Document 12: JP-A-8-133948
特許文献 13:特開 2003— 160463 Patent Document 13: Japanese Patent Laid-Open No. 2003-160463
特許文献 14:特開 2004— 339119 Patent Document 14: JP 2004-339119 A
特許文献 15:特開平 5— 310727 Patent Document 15: JP-A-5-310727
特許文献 16:US6767741 Patent Document 16: US6767741
非特許文献 1: Nature Rev. Drug. Discov. , 3, 383(2004) Non-Patent Document 1: Nature Rev. Drug. Discov., 3, 383 (2004)
非特許文献 2:Nature biotechnology, 19, Mar. 21, 184(2001) Non-Patent Document 2: Nature biotechnology, 19, Mar. 21, 184 (2001)
非特許文献 3: Nature biotechnology, 22, Nov. 11, 1393(2004) 非特許文献 4 :日薬理誌， 83, 207 (1984) Non-Patent Document 3: Nature biotechnology, 22, Nov. 11, 1393 (2004) Non-Patent Document 4: Pharmacological Journal of Japan, 83, 207 (1984)
非特許文献 5 :歯学， 75 (5) , 985 (1987)  Non-Patent Document 5: Dentistry, 75 (5), 985 (1987)
非特許文献 6 :歯科医学， 52 (6) , 745 (1989)  Non-Patent Document 6: Dental Medicine, 52 (6), 745 (1989)
発明の開示  Disclosure of the invention
発明が解決しょうとする課題  Problems to be solved by the invention
[0009] 動物細胞を培養し、該細胞力 物質を生産させる方法にぉ 、て、生産される物質 の生産性を向上するための方法が求められている。 [0009] A method for improving the productivity of a substance to be produced is required in addition to a method for culturing animal cells and producing the cellular force substance.
課題を解決するための手段  Means for solving the problem
[0010] 本発明は、以下の（1)〜（27)に関する。 The present invention relates to the following (1) to (27).
(1)以下の（a)〜（e)力も選ばれる少なくとも 1つの物質を添加した培地中で、物質を 生産する能力を有する動物細胞を培養し、培養物中に該物質を生成蓄積させ、該培 養物から該物質を採取すること、を特徴とする物質の製造方法。  (1) The following (a) to (e) culturing animal cells having the ability to produce a substance in a medium supplemented with at least one substance selected also for force, and producing and accumulating the substance in the culture, A method for producing a substance, comprising collecting the substance from the culture.
(a)リグナン類  (a) Lignans
(b)フラボノイド類  (b) Flavonoids
(c)ヒストン脱ァセチルイ匕酵素阻害剤  (c) Histone deacetylase inhibitor
(d)テルぺノイド類  (d) Terpenoids
(e)コウジ酸またはその誘導体  (e) Kojic acid or its derivatives
[0011] (2)テルぺノイド類がカンファである、（1)に記載の方法。  [0011] (2) The method according to (1), wherein the terpenoid is camphor.
(3)動物細胞が哺乳類に属する動物細胞である、（1)または（2)に記載の方法。 (3) The method according to (1) or (2), wherein the animal cell is an animal cell belonging to a mammal.
(4)哺乳類に属する動物が霊長類または齧歯類である、 (3)に記載の方法。 (4) The method according to (3), wherein the animal belonging to the mammal is a primate or a rodent.
(5)動物細胞がミエローマ細胞または卵巣細胞、あるいはこれらの細胞に由来する 細胞である、 (1)〜 (4)のいずれか 1項に記載の方法。  (5) The method according to any one of (1) to (4), wherein the animal cell is a myeloma cell or ovary cell, or a cell derived from these cells.
(6)物質がペプチドである、 (1)〜（5)の 、ずれか 1項に記載の方法。  (6) The method according to any one of (1) to (5), wherein the substance is a peptide.
[0012] (7)動物細胞がペプチドをコードする遺伝子を含有するベクターが導入された形質 転換細胞である、 (6)に記載の方法。  [0012] (7) The method according to (6), wherein the animal cell is a transformed cell into which a vector containing a gene encoding a peptide is introduced.
(8)ペプチドが糖蛋白質である、 (6)または（7)に記載の方法。  (8) The method according to (6) or (7), wherein the peptide is a glycoprotein.
(9)糖蛋白質が抗体である、（8)に記載の方法。  (9) The method according to (8), wherein the glycoprotein is an antibody.
[0013] (10)以下の（a)〜（e)から選ばれる少なくとも 1つの物質を添カ卩した培地中で、物質 を生産する能力を有する動物細胞を培養し、該動物細胞より生産される物質の細胞 あたりの生産性を向上させた後、培養物中に該物質を生成蓄積させ、該培養物から 該物質を採取することを特徴とする物質の製造方法。 [0013] (10) In a medium supplemented with at least one substance selected from the following (a) to (e): After culturing animal cells having the ability to produce and improving the productivity per cell of the substance produced from the animal cell, 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 being collected.
(a)リグナン類  (a) Lignans
(b)フラボノイド類  (b) Flavonoids
(c)ヒストン脱ァセチルイ匕酵素阻害剤  (c) Histone deacetylase inhibitor
(d)テルぺノイド類  (d) Terpenoids
(e)コウジ酸またはその誘導体  (e) Kojic acid or its derivatives
(11)テルぺノイド類がカンファである、 (10)に記載の方法。  (11) The method according to (10), wherein the terpenoid is camphor.
(12)動物細胞が哺乳類に属する動物細胞である、（10)または（11)に記載の方法。  (12) The method according to (10) or (11), wherein the animal cell is an animal cell belonging to a mammal.
[0014] (13)哺乳類に属する動物が霊長類または齧歯類である、（12)に記載の方法。 [0014] (13) The method according to (12), wherein the animal belonging to the mammal is a primate or a rodent.
(14)動物細胞がミエローマ細胞または卵巣細胞、あるいはこれらの細胞に由来する 細胞である、（10)〜（13)のいずれか 1項に記載の方法。  (14) The method according to any one of (10) to (13), wherein the animal cell is a myeloma cell or ovary cell, or a cell derived from these cells.
(15)物質がペプチドである、 (10)〜（14)のいずれか 1項に記載の方法。  (15) The method according to any one of (10) to (14), wherein the substance is a peptide.
[0015] (16)動物細胞がペプチドをコードする遺伝子を含有するベクターが導入された形質 転換細胞である、（15)に記載の方法。  [0015] (16) The method according to (15), wherein the animal cell is a transformed cell into which a vector containing a gene encoding a peptide is introduced.
(17)ペプチドが糖蛋白質である、（15)または（16)に記載の方法。  (17) The method according to (15) or (16), wherein the peptide is a glycoprotein.
[0016] (18)糖蛋白質が抗体である、（17)に記載の方法。 [0016] (18) The method according to (17), wherein the glycoprotein is an antibody.
(19)以下の（a)〜（e)力も選ばれる少なくとも 1つの物質を添加した培地中で、物質 を生産する能力を有する動物細胞を培養することを特徴とする、該動物細胞より生産 される物質の細胞あたりの生産性を向上させる方法。  (19) An animal cell having the ability to produce a substance is cultured in a medium supplemented with at least one substance selected from the following (a) to (e) force: To improve the per-cell productivity of a certain substance.
(a)リグナン類  (a) Lignans
(b)フラボノイド類  (b) Flavonoids
(c)ヒストン脱ァセチルイ匕酵素阻害剤  (c) Histone deacetylase inhibitor
(d)テルぺノイド類  (d) Terpenoids
(e)コウジ酸またはその誘導体  (e) Kojic acid or its derivatives
(20)テルぺノイド類がカンファである、 (19)に記載の方法。  (20) The method according to (19), wherein the terpenoid is camphor.
[0017] (21)動物細胞が哺乳類に属する動物細胞である、（19)または（20)に記載の方法。 (22)哺乳類に属する動物が霊長類または齧歯類である、 (21)に記載の方法。[0017] (21) The method according to (19) or (20), wherein the animal cell is an animal cell belonging to a mammal. (22) The method according to (21), wherein the animal belonging to the mammal is a primate or a rodent.
(23)動物細胞がミエローマ細胞または卵巣細胞、あるいはこれらの細胞に由来する 細胞である、（19)〜（22)のいずれか 1項に記載の方法。 (23) The method according to any one of (19) to (22), wherein the animal cell is a myeloma cell or ovary cell, or a cell derived from these cells.
[0018] (24)物質がペプチドである、（19)〜（23)のいずれか 1項に記載の方法。  [0018] (24) The method according to any one of (19) to (23), wherein the substance is a peptide.
(25)動物細胞がペプチドをコードする遺伝子を含有するベクターが導入された形質 転換細胞である、 (24)に記載の方法。  (25) The method according to (24), wherein the animal cell is a transformed cell into which a vector containing a gene encoding a peptide is introduced.
(26)ペプチドが糖蛋白質である、 (24)または（25)に記載の方法。  (26) The method according to (24) or (25), wherein the peptide is a glycoprotein.
(27)糖蛋白質が抗体である、 (26)に記載の方法。  (27) The method according to (26), wherein the glycoprotein is an antibody.
発明の効果  The invention's effect
[0019] 本発明によると、リグナン類、フラボノイド類、ヒストン脱ァセチルイ匕酵素阻害剤、テ ルぺノイド類、カンファ、コウジ酸またはその誘導体力 選ばれる少なくとも 1つの物質 を添加した培地中で動物細胞を培養することを特徴とする物質の製造方法、およびリ ダナン類、フラボノイド類、ヒストン脱ァセチルイ匕酵素阻害剤、テルぺノイド類、カンフ ァ、コウジ酸またはその誘導体力 選ばれる少なくとも 1つの物質を添加した培地を用 いることを特徴とする、物質の細胞あたりの生産性を向上させる方法が提供される。 図面の簡単な説明  [0019] According to the present invention, animal cells in a medium to which at least one substance selected from lignans, flavonoids, histone deacetylase inhibitors, terpenoids, camphor, kojic acid or their derivatives are added. And a method for producing a substance characterized by culturing lydanan, flavonoids, histone deacetylase inhibitor, terpenoids, camphor, kojic acid or a derivative thereof, at least one substance selected from There is provided a method for improving the productivity per cell of a substance, characterized by using an added medium. Brief Description of Drawings
[0020] [図 1]図 1は、 Ms704— CD20株用いて三角フラスコにおいてフエドバッチ培養を行 つたときの、培養開始から 14日目における細胞あたりの比生産速度 (pgZ細胞 Z日） を示した図である。  [0020] [Fig. 1] Fig. 1 shows the specific production rate per cell (pgZ cell Z day) on the 14th day from the start of the culture when the Ms704-CD20 strain was used in a batch flask in an Erlenmeyer flask. FIG.
[0021] [図 2]図 2は、 Ms705-pKAN— ΑΤΠΙ株を用いて三角フラスコにおいてフエドバッチ 培養を行ったときの、培養開始から 14日目における細胞あたりの比生産速度 (pgZ 細胞 Z日）を示した図である。図中國は各濃度でカンファを添加した培養を、□はコ ントロール培養をそれぞれ示す。  [0021] [Figure 2] Figure 2 shows the specific production rate per cell (pgZ cell Z day) on the 14th day from the start of the culture when fed batch culture was performed in an Erlenmeyer flask using Ms705-pKAN-— strain. FIG. In the figure, China indicates the culture with camphor added at each concentration, and □ indicates the control culture.
[0022] [図 3]図 3は、 Ms705-pKAN— ΑΤΠΙ株を用いて三角フラスコにおいてフエドバッチ 培養を行ったときの累積生細胞密度 (細胞/ mL X日）に対する生産された AT— ΠΙ濃 度（mg/L)を示した図である。図中 Xはコントロール培養を、口は 0. 12mmolZL、 ♦は 0. 23mmol/L、△は 0. 39mmol/L,拳は 0. 55mmol/Lでカンファを添カロ した培養をそれぞれ示す。 [0023] [図 4]Ms704ZCD20株を用いて三角フラスコにお!/、てフエドバッチ培養を行ったと きの細胞あたりの比生産速度 (pgZ細胞 Z日）を示した図である。図中國はカンファ を添カ卩した培養を、□はコントロール培養をそれぞれ示す。 [0022] [Fig. 3] Fig. 3 shows the concentration of AT- に 対 す る produced against the cumulative viable cell density (cells / mL x day) when fed-batch culture was performed in Erlenmeyer flasks using Ms705-pKAN-ΑΤΠΙ strain. It is the figure which showed (mg / L). In the figure, X indicates control culture, mouth indicates 0.12 mmol ZL, ♦ indicates 0.23 mmol / L, Δ indicates 0.39 mmol / L, and fist indicates 0.55 mmol / L with camphor. [0023] [Fig. 4] A graph showing the specific production rate per cell (pgZ cell Z day) when Ms704ZCD20 strain was used in a Erlenmeyer flask! In the figure, China indicates culture with camphor and □ indicates control culture.
[0024] [図 5]709 LCA—500G株を用いて三角フラスコにおいてフエドバッチ培養を行った ときの細胞あたりの比生産速度 (pgZ細胞 Z日）を示した図である。図中國はカンフ ァを添加した培養を、□はコントロール培養をそれぞれ示す。  [0024] FIG. 5 is a graph showing the specific production rate per cell (pgZ cell Z days) when a fed batch culture was performed in an Erlenmeyer flask using the 709 LCA-500G strain. In the figure, China represents the culture with the addition of camphor, and □ represents the control culture.
[0025] [図 6]Ms704ZCD20株を用いて三角フラスコにお!/、てフエドバッチ培養を行ったと きの、生細胞密度の経時変化を示した図である。図中▲は培養開始と同時にカンフ ァを添加した培養を、參は培養開始から 3日目にカンファを添加した培養を、國は培 養開始から 5日目にカンファを添加した培養を、♦は培養開始から 7日目にカンファ を添加した培養を、 Xは培養開始から 9日目にカンファを添力！]した培養を、 *は培養 開始から 11日目にカンファを添加した培養を、〇はコントロール培養をそれぞれ示 す。  [0025] FIG. 6 is a graph showing changes in the density of viable cells with time when! /, And Fed batch culture were performed on Erlenmeyer flasks using Ms704ZCD20 strain. In the figure, ▲ indicates the culture with the addition of camphor at the start of the culture, 參 indicates the culture with the addition of camphor on the third day from the start of culture, and Kuni indicates the culture with the addition of camphor on the fifth day from the start of the culture. Shows the culture with camphor added on the 7th day from the start of culture, and X adds camphor on the 9th day after the start of culture! ] Indicates a culture with camphor added on day 11 from the start of culture, and ◯ indicates a control culture.
[0026] [図 7]Ms704ZCD20株を用いて三角フラスコにお!/、てフエドバッチ培養を行ったと きの細胞あたりの比生産速度 (pgZ細胞 Z日）を示した図である。図中の國カンファ を添加した時期毎の培養を、□はコントロール培養をそれぞれ示す。  [0026] FIG. 7 is a graph showing the specific production rate per cell (pgZ cell Z days) when! /, Fed-batch culture was performed in an Erlenmeyer flask using Ms704ZCD20 strain. In the figure, □ indicates the culture at each time when the country camphor was added, and □ indicates the control culture.
[0027] [図 8]図 8は、 709 LCA—500G株を用いて三角フラスコにおいて ImmolZLの濃 度でコウジ酸を添加した培地でのフエドバッチ培養を行ったときの、培養開始から 15 日目における、生細胞密度の経時変化を示した図である。図中ロはコウジ酸を培養 開始と同時に、◊は培養開始から 3日目に、 は培養開始から 5日目に添加した培 養をそれぞれ示し、參はコントロール培養を示す。  [0027] [Fig. 8] Fig. 8 is a graph showing 15 days from the start of culture when 709 LCA-500G strain was fed batch culture in an Erlenmeyer flask and supplemented with kojic acid at a concentration of ImmolZL. FIG. 3 is a graph showing a change with time in the density of living cells. In the figure, (b) indicates the culture added with kojic acid at the same time as the start of cultivation.
[0028] [図 9]図 9は、 709 LCA—500G株を用いて三角フラスコにおいてフエドバッチ培養 を行ったときの細胞あたりの比生産速度 (pgZ細胞 Z日）を示した図である。図中の 枠は、左力 コントロール、培養開始と同時、 3日目および 5日目にコウジ酸を添加し た培養をそれぞれ示す。コウジ酸を添加した培養において、白抜きのカラムはコウジ 酸を 0. 5mmolZLで黒塗りのカラムはコウジ酸を 1. OmmolZLで添カ卩した培養を それぞれ示す。  [0028] [Fig. 9] Fig. 9 shows the specific production rate (pgZ cell Z day) per cell when fed batch culture was performed in an Erlenmeyer flask using the 709 LCA-500G strain. The frame in the figure shows the left force control, the culture with addition of kojic acid on the 3rd and 5th days at the same time as the start of the culture. In the culture in which kojic acid was added, the white column represents the culture in which kojic acid was added at 0.5 mmol ZL and the black column was added with kojic acid at 1. O mmol ZL.
発明を実施するための最良の形態 [0029] 本発明は、リグナン類、フラボノイド類、ヒストン脱ァセチルイ匕酵素阻害剤、テルぺノ イド類、コウジ酸またはその誘導体力も選ばれる少なくとも 1つの物質を添加した培地 中で動物細胞を培養することを特徴とする物質の製造方法、およびリグナン類、フラ ボノイド類、ヒストン脱ァセチルイ匕酵素阻害剤、テルぺノイド類、コウジ酸またはその誘 導体力も選ばれる少なくとも 1つの物質を添加した培地中で動物細胞を培養すること を特徴とする、物質の細胞あたりの生産性を向上させる方法に関する。 BEST MODE FOR CARRYING OUT THE INVENTION [0029] The present invention cultivates animal cells in a medium to which at least one substance selected also for lignans, flavonoids, histone deacetylase inhibitors, terpenoids, kojic acid or derivatives thereof is added. And in a medium containing lignans, flavonoids, histone deacetylase inhibitors, terpenoids, kojic acid, or at least one substance whose inductive capacity is also selected. It is related with the method of improving the productivity per cell of a substance characterized by cultivating an animal cell in (3).
[0030] 本発明における培地に添加するリグナン類、フラボノイド類、ヒストン脱ァセチルイ匕 酵素阻害剤、テルぺノイド類には、それらの物質の配糖体などの類縁体および誘導 体も包含される。  [0030] The lignans, flavonoids, histone deacetylase enzyme inhibitors and terpenoids added to the medium in the present invention also include analogs and derivatives such as glycosides of these substances.
リグナン類とは、フエ-ルプロパノイド 2分子が β炭素間で結合した [C6-C3]を基本 構造とする一群の化合物をいう。 ）8炭素以外の炭素、酸素間で結合したネオリグナン (neolignan)、 3分子のフエ-ルプロパノイドが結合したセスキリグナン（sesquilignan)、 4分子が結合したジリグナン (dilignan)等もリグナン類に包含される。本発明に用いら れるリグナン類としては、リグナン類であれば特に制限はないが、具体的には、ポドフ イロトキシン、セサミン、セサミノール、セサモーノレ、セサモリノール、セサモリン、シザ ンドリン、ゴミシン A、マグノロール、ホオノキオール、フエ-ルプロパノイド、ゴミシン A などがあげられ、セサモールが好適に用いられる。 Lignans are a group of compounds whose basic structure is [C 6 -C 3 ], in which two molecules of phenylpropanoid are bonded between β-carbons. ) Neligignan bonded between carbons other than 8 carbons and oxygen, sesquilignan bonded with 3 molecule propanoids, dilignan bonded with 4 molecules, etc. are also included in the lignans. The lignans used in the present invention are not particularly limited as long as they are lignans. Specifically, podophyllotoxin, sesamin, sesaminol, sesamonole, sesamorinol, sesamolin, cisandrin, trashin A, magnolol, Examples include honokiol, phenolpropanoid, and trashin A. Sesamol is preferably used.
[0031] フラボノイド類は、植物に広く含まれる色素成分、ポリフエノール混合物群の総称で あり、 2つのフエニル基が 3つの炭素を介して結合した [C6-C3_C6]を基本構造とする 一群の化合物をいう。本発明に用いられるフラボノイド類としては、特に制限はないが 具体的にはフラバノン、フラボン、カルコン、フラバノール (カテキン)、フラバノノール、 フラボノール、オーロン、フラバン- 3,4-ジオール (ロイコアントシアン)、イソフラボン、ィ ソフラボノイドなどがあげられ、フラボン、イソフラボノイドなどが好適に用いられる。フ ラボンとしては、ノレテオリン、ァカセチン、クリシンまたはァピゲニンなどがあげられる 力 ルテオリン、ァカセチンなどが好適に用いられる。イソフラボノイドとしては、ダイゼ イン、ゲニスチンまたはグリシティンなどがあげられる力 ダイゼインが好適に用いら れる。 [0031] Flavonoids are a general term for a mixture of pigment components and polyphenols widely contained in plants. The basic structure is [C 6 -C 3 _C 6 ] in which two phenyl groups are bonded via three carbons. Yes Refers to a group of compounds. The flavonoids used in the present invention are not particularly limited, but specifically, flavanone, flavone, chalcone, flavanol (catechin), flavonol, flavonol, aurone, flavan-3,4-diol (leucoanthocyan), isoflavone, Examples include isoflavonoids, and flavones and isoflavonoids are preferably used. As flavones, strong luteolin, acacetine, and the like, which are exemplified by noreteolin, acacetin, chrysin or apigenin, are preferably used. As the isoflavonoid, force daidzein such as daidzein, genistin or glycitin is preferably used.
[0032] 本発明に用いられるヒストン脱ァセチルイ匕酵素阻害剤としては、バルプロ酸、バル プロ酸ナトリウム、酪酸ナトリウム、スべロイドア-リド、アビシジントラポキシン、ヒドロキ サム酸などがあげられる力 バルプロ酸ナトリウムが好適に用いられる。 [0032] Examples of the histone deacetylase inhibitor used in the present invention include valproic acid and valproic acid. Sodium valproate, which includes sodium proate, sodium butyrate, sveroidalide, avicidin trapoxin, hydroxamic acid and the like, is preferably used.
テルぺノイド類は、イソプレンの重合したィ匕合物である。テルぺノイド類としては、モ ノテルペン (C )、セスキテルペン (C )、ジテルペン (C )、セスタテルペン (C )、トリテ  Terpenoids are isoprene-polymerized compounds. Terpenoids include monoterpenes (C), sesquiterpenes (C), diterpenes (C), sesterterpenes (C), trite
10 15 20 25 ルペン ),テトラテルペン (C )またはそれらの配糖体などがあげられる。  10 15 20 25 Rupen), tetraterpene (C) or their glycosides.
30 40  30 40
モノテルペンとしては、ゲラ-オール、ネロール、リナロール、シトラール (ゲラ -ァー ル)、シネオール、シトロネロール、メントール、リモネン、テルビネロール、力ノレボン、ョ ノン、ッヨン、カンファ、ボルネオール、ァネトール、オイゲノールなどがあげられる。セ スキテルペンとしては、フアルネソール、ネロリドール、幼若ホルモン、フムレン、カリオ フィレン、エレメン、カジノール、カジネン、ッチンなどがあげられる。ジテルペンとして は、ゲラ-ルゲラ-オール、フィトール、ァビエチン酸、ピマラジェン、ダフネトキシン、 タキノール、センプレンなどがあげられる。トリテルペンとしては、スクアレン、リモニン 、カメリアゲニン、ホパン、ラノステロール、サポニンなどがあげられる。テトラテルペン としては、カロテノイドなどがあげられる。配糖体としてはグリチルレチン酸およびその 誘導体などがあげられる。本発明に用いられるテルぺノイド類はテルぺノイド類であ れば特に制限はないが、カンファ、サポニン、グリチルレチン酸およびその誘導体な どが好適に用いられる。サポニンとしては、オンジサポニン Gがあげられる。ダリチル レチン酸およびその誘導体としては、 OC グリチルレチン酸、 β グリチルレチン酸、 a グリチルレチン酸ステアリル、 13 グリチルレチン酸ステアリル、 at グリチルレ チン酸ピリドキシン、 j8—グリチルレチン酸ピリドキシン、 a グリチルレチン酸グリセ リン、 13 グリチルレチン酸グリセリン、 3—サクシ-ルォキシグリチルレチン酸ニナトリ ゥム等のグリチルレチン酸誘導体、 18 α グリチルリチン酸、 18 j8—グリチルリチン 酸、 18 α グリチルリチン酸メチルエステル、 18 j8—グリチルリチン酸メチルエステ ル、 18 α グリチルリチン酸卜リナ卜リウム、 18 α グリチルリチン酸モノカリウム、 18 a グリチルリチン酸ジカリウム、 18 a グリチルリチン酸モノアンモ-ゥム、 18 j8— グリチルリチン酸トリナトリウム、 18 |8—グリチルリチン酸モノカリウム、 18 |8—ダリチル リチン酸ジカリウム、 18 j8—グリチルリチン酸モノアンモ-ゥム等のグリチルリチン酸 誘導体があげられる力 18 α グリチルレチン酸が好適に用いられる。 [0034] 本発明における培地に添加するモノテルペンのうち、カンファ (カンファ以外にも、 ショウノウ、カンファー、カンフル、カンファァなどとも称される。 )としては、カンファおよ びその類縁体があげられる。カンファおよびその類縁体は、 d体、 1体、 dl体のいずれ の光学異性体でもよぐカンファの誘導体でもよい。また、これらを含有した物質、例 えば精油を用いることもできる。 Monoterpenes include gala-ol, nerol, linalool, citral (gera-al), cineol, citronellol, menthol, limonene, terbinerol, force norebon, nonon, gyon, camphor, borneol, anetor, eugenol. It is done. Examples of sesquiterpenes include fuarnesol, nerolidol, juvenile hormone, humulene, cariophyllene, elemental, casinodal, kadinene, and chin. Examples of diterpenes include gerageruger-ol, phytol, abietic acid, pimaragen, daphnetoxin, taquinol, and semprene. Triterpenes include squalene, limonin, cameliagenin, hopane, lanosterol, saponin and the like. Examples of tetraterpenes include carotenoids. Examples of glycosides include glycyrrhetinic acid and its derivatives. The terpenoid used in the present invention is not particularly limited as long as it is a terpenoid, but camphor, saponin, glycyrrhetinic acid and derivatives thereof are preferably used. Onjisaponin G is an example of saponin. Daricylretinoic acid and its derivatives include OC glycyrrhetinic acid, β-glycyrrhetinic acid, a stearyl glycyrrhetinate, 13 stearyl glycyrrhetinate, pyridoxine glycyrrhetinate, j8-glycyrrhetinic acid pyridoxine, a glycerin glycyrrhetinate, glycerin 13 glycyrrhetinate, Glycyrrhetinic acid derivatives such as 3-succinoxy glycyrrhetinic acid ninatrium, 18 α-glycyrrhizic acid, 18 j8-glycyrrhizic acid, 18 α-glycyrrhizic acid methyl ester, 18 j8-glycyrrhizic acid methyl ester, 18 α-glycyrrhizic acid linna卜, 18 α monopotassium glycyrrhizinate, 18 a dipotassium glycyrrhizinate, 18 a monoammonium glycyrrhizinate, 18 j8—trisodium glycyrrhizinate, 18 | 8—glycyrrhizin Monopotassium phosphate, 18 | 8- Darichiru ricin dipotassium, 18 j8- glycyrrhizinate Monoanmo - © beam force 18 alpha glycyrrhetinic acid glycyrrhizinate derivatives such as is preferably used. [0034] Among the monoterpenes added to the culture medium in the present invention, camphor (in addition to camphor, also referred to as camphor, camphor, camphor, camphor, etc.) includes camphor and its analogs. Camphor and its analogs may be either d-form, 1-form, dl-form optical isomers or camphor derivatives. In addition, substances containing these, such as essential oils, can also be used.
[0035] カンファの誘導体としては、例えば、カンファン酸およびその塩類、カンフェン、ショ ウノゥ酸およびその塩類、カンホルキノン、カンホルキノン 3—ォキシム、カンフォルス ルフォキシド、 10—力ンフォルスルフォ -ルォキサジリジン、カンフアスルホン酸およ びその塩、カンファ t—トシルヒドラゾン、またはケトピニック酸およびその塩類などがあ げられる。また、これらの誘導体のグルクロン酸包合体類、 3—ヒドロキシカンファ、 5 ーヒドロキシカンファまたは 8—ヒドロキシ， 9ーヒドロキシカンファなどの代謝物などが あげられる。  [0035] Examples of camphor derivatives include camphanic acid and its salts, camphene, succinic acid and its salts, camphorquinone, camphorquinone 3-oxime, camphorsulfoxide, 10-strong sulfo-loxaziridine, camphorsulfone Examples include acids and salts thereof, camphor t-tosylhydrazone, or ketopic acids and salts thereof. In addition, glucuronic acid conjugates of these derivatives, metabolites such as 3-hydroxycamphor, 5-hydroxycamphor, 8-hydroxy, 9-hydroxycamphor, and the like can be mentioned.
[0036] 本発明における培地に添加するコウジ酸は、下記のような構造式で表される化合物 であるが、本発明に用いられるコウジ酸としては、下記化合物の他、下記化合物の塩 や配糖体なども包含する。  [0036] Kojic acid to be added to the medium in the present invention is a compound represented by the following structural formula. Examples of kojic acid used in the present invention include the following compounds, salts and salts of the following compounds. Also includes saccharides.
[化 1]  [Chemical 1]
Figure imgf000011_0001
Figure imgf000011_0001
[0037] 塩としては、例えば、例えばアルカリ金属塩 (例えば、ナトリウム塩、カリウム塩、セシ ゥム塩など）、アルカリ土類金属塩 (例えば、カルシウム塩、マグネシウム塩など）など の金属塩、アンモ-ゥム塩、有機塩基との塩 (例えば、トリメチルァミン塩、トリェチル アミン塩、ピリジン塩、ピコリン塩、ェタノ一ルァミン塩、トリェタノ一ルァミン塩、ジシク 口へキシルァミン塩、 N, N，一ジベンジルエチレンジァミン塩など）、アミノ酸との塩（ 例えば、アルギニン塩、ァスパラギン酸塩、グルタミン酸塩など）などがあげられる。 [0037] Examples of the salt include metal salts such as alkali metal salts (for example, sodium salts, potassium salts, and cesium salts), alkaline earth metal salts (for example, calcium salts, magnesium salts, and the like), ammonia -Um salt, salt with organic base (for example, trimethylamine salt, triethylamine salt, pyridine salt, picoline salt, ethano-lamine salt, trihetano-lamine salt, dioxyhexylamine salt, N, N, Benzylethylenediamine salt), salts with amino acids ( For example, arginine salt, aspartate, glutamate and the like).
[0038] 配糖体としては、グルコースが結合した配糖体である、コウジ酸モノダルコサイド、コ ウジ酸ジダルコサイド、コウジ酸トリダルコサイド、コウジ酸テトラダルコサイド (特開平 0 7— 236496)およびコウジ酸 5— 0— a—D—ダルコビラノシド（特開平 05— 07838 3)、フルクトースが結合したコウジ酸フラクトシド類 (特開平 05— 078383、特開平 10 — 099091)、ガラクトースが結合したコウジ酸ガラクトシド（特開平 08— 134090)な どがあげられる。  [0038] Examples of glycosides include glucose-bound glycosides, kojic acid monodalcoside, kojic acid tridarcoside, kojic acid tridarcoside (Japanese Patent Laid-Open No. 07-2336496) and Kojic acid 5-0-a-D-Dalcobilanoside (JP 05-078383), fructose-bound kojic acid fructosides (JP 05-0778383, JP 10-099091), galactose-bound kojic acid galactoside ( JP-A-08-134090).
[0039] 本発明におけるコウジ酸誘導体としては、例えば、コウジ酸エステル誘導体 (特公 昭 60— 9722号、特公昭 61— 60801号、特開 2000— 344760、特開 2003— 155 283)、コウジ酸エーテル誘導体 (特開平 3— 14508号）、 2—位がモノまたはジヒドロ キシ安息香酸で置換されたコウジ酸誘導体 (特開平 07— 188206)、並びにコウジ酸 の 2量体 (特開平 05— 310727)などがあげられる。コウジ酸誘導体の塩や配糖体も 本発明のコウジ酸誘導体に包含される。  As the kojic acid derivative in the present invention, for example, kojic acid ester derivatives (Japanese Patent Publication Nos. 60-9722, 61-60801, JP 2000-344760, JP 2003-155 283), Kojic acid Ether derivatives (JP-A-3-14508), kojic acid derivatives substituted at the 2-position with mono- or dihydroxybenzoic acid (JP-A-07-188206), and dimers of kojic acid (JP-A 05-310727) Etc. Kojic acid derivative salts and glycosides are also included in the kojic acid derivative of the present invention.
[0040] 以下、コウジ酸またはその誘導体、それらの塩や配糖体を総称して、コウジ酸類と [0040] Hereinafter, kojic acid or its derivatives, their salts and glycosides are collectively referred to as kojic acids.
Β§ίί己す。。 Β§ίίMe .
[0041] 本発明に用いられる培地は、動物細胞の培養に用いることができればいかなるもの でも良ぐ血清含有培地、無血清培地、培地中に動物蛋白質由来物を含まない培地 、無蛋白培地などがあげられるが、無血清培地、無蛋白培地が好ましい。  [0041] The medium used in the present invention may be any serum-containing medium, serum-free medium, medium not containing animal protein-derived medium, protein-free medium, etc., as long as it can be used for animal cell culture. A serum-free medium and a protein-free medium are preferable.
本発明の方法にぉ 、て用 、られる、通常の動物細胞の培養に用 、られる基礎培地 としては、例えば RPMI1640培地 [The Journal of the American Medical Association, 199, 519 (1967) ]、 Eagleの MEM培地 [Science, 122, 501 (19 52) ]、ダルベッコ改変 MEM (DMEM)培地 [Virology, 8, 396 (1959) ]、 199培 地 [Proceeding of the Society for the Biological Medicine, 73, 1 (19 50) ]、 F12培地（LTI社製） [Proc. Natl. Acad. Sci. USA, 53, 288 (196 5) ]、イスコフ改変ダルベッコ培地（IMDM培地） [J. Experimental Medicine,丄 47, 923 (1978) ]、 EX— CELL™302培地 (JRH社製）またはこれらの改変培地や 混合培地などがあげられる力 好ましくは RPMI1640培地、 DMEM、 F12培地、 I MDMおよび EX—CELL™302培地などが用いられる。 [0042] 血清含有培地としては、上記の基礎培地に、ゥシ、ゥマ、魚などの動物血清を適当 量、通常は 5〜10%前後を添カ卩したものが用いられる。 Examples of basal media used for normal animal cell culture used in the method of the present invention include RPMI1640 medium [The Journal of the American Medical Association, 199, 519 (1967)], Eagle's MEM Medium [Science, 122, 501 (19 52)], Dulbecco's modified MEM (DMEM) medium [Virology, 8, 396 (1959)], 199 medium [Proceeding of the Society for the Biological Medicine, 73, 1 (1950 ], F12 medium (manufactured by LTI) [Proc. Natl. Acad. Sci. USA, 53, 288 (196 5)], Iskov modified Dulbecco medium (IMDM medium) [J. Experimental Medicine, 丄 47, 923 (1978) )], EX—CELL ™ 302 medium (manufactured by JRH) or their modified or mixed medium, preferably RPMI1640 medium, DMEM, F12 medium, I MDM and EX-CELL ™ 302 medium . [0042] As the serum-containing medium, a medium containing the above basal medium supplemented with an appropriate amount of animal serum such as horsetail, horse, fish, etc., usually around 5 to 10%, is used.
無血清培地には、必要に応じて動物細胞の生育に必要な栄養因子、生理活性物 質などを添加する。これらの添加物は、培養前に予め培地に含有させることが好まし い。  To the serum-free medium, add nutritional factors and physiologically active substances necessary for the growth of animal cells as necessary. These additives are preferably contained in the medium in advance before culturing.
栄養因子としては、グルコース、アミノ酸、ビタミンなどがあげられる。  Examples of nutrient factors include glucose, amino acids, vitamins and the like.
[0043] アミノ酸としては、 L ァラニン、 L アルギニン、 L ァスパラギン、 L ァスパラギ ン酸、 L シスチン、 L グルタミン酸、 L グルタミン、グリシン、 L ヒスチジン、 L- イソロイシン、 L ロイシン、 L リジン、 L—メチォニン、 L フエ二ルァラニン、 L プ 口リン、 Lーセリン、 Lースレオニン、 L—トリプトファン、 Lーチロシン、 L パリンなどが あげられ、 1種または 2種以上組み合わせて用いられる。 [0043] 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-leucine, L-lysine, L-methionine, L Examples include phenylalanine, L-pulin, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-parin, which can be used alone or in combination.
[0044] ビタミンとしては、 d—ピオチン、 D—パントテン酸、コリン、葉酸、 myo—イノシトール 、ナイァシンアミド、ピリドキサール、リボフラビン、チアミン、シァノコバラミン、 DL— α トコフエロールなどがあげられ、 1種または 2種以上組み合わせて用いられる。 生理活性物質としては、インシュリン、トランスフェリン、血清アルブミン、増殖因子を 含む血清分画物などがあげられる。 [0044] Examples of vitamins include d-piotine, D-pantothenic acid, choline, folic acid, myo-inositol, niacinamide, pyridoxal, riboflavin, thiamine, cyanocobalamin, DL-α tocopherol, and one or two of them Used in combination. Examples of physiologically active substances include insulin, transferrin, serum albumin, and serum fractions containing growth factors.
[0045] 動物由来原料を含まない培地としては、血清アルブミンや血清分画物などの動物 由来材料の代わりに、遺伝子組換え法で製造された生理活性物質や、加水分解物 または動物由来原料を含まない脂質を添加した培地があげられる。 [0045] As a medium not containing animal-derived materials, physiologically active substances produced by genetic recombination methods, hydrolysates, or animal-derived materials may be used instead of animal-derived materials such as serum albumin and serum fractions. Examples thereof include a medium supplemented with lipids not contained.
加水分解物としては、大豆、小麦、米、えんどう豆、綿実または酵母抽出物などの 加水分解物などがあげられる。  Examples of the hydrolyzate include hydrolysates such as soybean, wheat, rice, peas, cottonseed or yeast extract.
[0046] 脂質としては、コレステロール、リノール酸、リノレイン酸などがあげられる。 [0046] Examples of lipids include cholesterol, linoleic acid, and linolenic acid.
無蛋白培地としては、 ADPF培地 (Animal derived protein free medium ;H yClone社製)、 CD— Hybridoma培地（インビトロジェン社製）、 CD— CHO培地（ィ ンビトロジェン社製)などがあげられる。  Examples of the protein-free medium include ADPF medium (Animal derived protein free medium; HyClone), CD-Hybridoma medium (Invitrogen), CD-CHO medium (Invitrogen), and the like.
長期間または高密度で培養を行う場合には、アミノ酸類およびビタミン類を高濃度 に含有した培地、例えば RPMI1640培地、 DMEM培地および F12培地を 1 : 1 : 1 の比率で混合した培地、 DMEM培地および F 12培地を 1： 1の比率で混合した培地 、ノ、イブリドーマ SFM培地 (インビトロジェン社製)などが好適に用いられる。 When culturing for a long period of time or at a high density, a medium containing a high concentration of amino acids and vitamins, such as a medium in which RPMI1640 medium, DMEM medium and F12 medium are mixed at a ratio of 1: 1: 1, DMEM medium And F 12 medium mixed at a ratio of 1: 1 , NO, and hybridoma SFM medium (manufactured by Invitrogen) are preferably used.
[0047] リグナン類、フラボノイド類、ヒストン脱ァセチルイ匕酵素阻害剤、テルぺノイド類、コゥ ジ酸類力ゝら選ばれる少なくとも 1つの物質を培地中へ添加する場合の濃度は、通常 1 0〜3000 μ mol/ 好ましくは 30〜2500 μ mol/ さらに好ましくは 50〜2200 μ mo\/ 特に好ましくは 550 μ molZLであるが、培養に用いる動物細胞の種類 、生産する物質の種類、リグナン類、フラボノイド類、ヒストン脱ァセチルイ匕酵素阻害 剤、テルぺノイド類、コウジ酸類力も選ばれる少なくとも 1つの物質の添カ卩時期または 添加物質の種類などにより適宜選択することができる。 [0047] The concentration in the case where at least one substance selected from lignans, flavonoids, histone deacetylase inhibitors, terpenoids, and kojic acids is added to the medium is usually 10 to 3000. μmol / Preferably 30-2500 μmol / More preferably 50-2200 μmo \ / Especially preferably 550 μmolZL, but the type of animal cells used for culture, the type of substance produced, lignans, flavonoids In addition, histone deacetylase inhibitor, terpenoids, and kojic acid can also be selected as appropriate according to the timing of addition of the at least one substance or the kind of additive.
[0048] リグナン類、フラボノイド類、ヒストン脱ァセチルイ匕酵素阻害剤、テルぺノイド類、コゥ ジ酸類力 選ばれる物質は培地に単独または複数の物質を同時にまたは異なる時 期に添加しても、複数の物質をあら力じめ混合して添加しても良い。また、リグナン類 、フラボノイド類、ヒストン脱ァセチルイ匕酵素阻害剤、テルぺノイド類、コウジ酸類から 選ばれる物質は単独または複数の物質を、細胞を播種する前の培地にあら力じめ添 カロしておいてもよいし、培養中の適当な時期に培地中に添加しても良い。さらに、複 数の物質を組み合わせる場合においては、動物細胞の培養において物質の生産性 を向上させることが知られている酪酸、レチノイン酸、コェンザィム Q10などを用いて も良い。 [0048] Lignans, flavonoids, histone deacetylase inhibitors, terpenoids, kojic acid power The substance selected may be a single substance, or multiple substances may be added to the medium simultaneously or at different times. These substances may be added after being mixed. In addition, a substance selected from lignans, flavonoids, histone deacetylase inhibitors, terpenoids, and kojic acids can be added to a medium before seeding the cells. It may be added to the medium at an appropriate time during the culture. Further, when a plurality of substances are combined, butyric acid, retinoic acid, and coenzyme Q10, which are known to improve the productivity of substances in animal cell culture, may be used.
[0049] 本発明に用いられる動物細胞としては、物質を生産する能力を有する動物細胞で あれば、例えば哺乳類、鳥類、は虫類、両生類、魚類および昆虫類などのいずれか に属する動物細胞など、いずれを用いてもよいが、哺乳類に属する動物細胞が好適 に用いられる。特に、ヒトまたはサルなどの霊長類に由来する動物細胞またはマウス、 ラットまたはハムスターなどの齧歯類に由来する動物細胞が好ましく用いられる。  [0049] The animal cell used in the present invention is any animal cell having the ability to produce a substance, such as an animal cell belonging to any of mammals, birds, reptiles, amphibians, fish, insects, etc. However, animal cells belonging to mammals are preferably used. In particular, animal cells derived from primates such as humans or monkeys or animal cells derived from rodents such as mice, rats or hamsters are preferably used.
[0050] 哺乳類に属する動物細胞としては、ミエローマ細胞、卵巣細胞、腎臓細胞、血球細 胞、子宮細胞、結合組織細胞、乳腺細胞または胚性網膜芽細胞、あるいはこれらの 細胞に由来する細胞などがあげられるが、特にミエローマ細胞または卵巣細胞、ある いはこれらの細胞に由来する細胞が好ましい。また、生産される物質が抗体である場 合は、ハイプリドーマなどの抗体産生細胞が好ましく用いられる。  [0050] Animal cells belonging to mammals include myeloma cells, ovarian cells, kidney cells, blood cells, uterine cells, connective tissue cells, mammary cells or embryonic retinoblasts, or cells derived from these cells. In particular, myeloma cells or ovary cells, or cells derived from these cells are preferred. In addition, when the substance to be produced is an antibody, antibody-producing cells such as hypridoma are preferably used.
[0051] 哺乳類に属する動物細胞としては、例えば、ヒト細胞株である HL— 60 (ATCC C CL— 240)、 HT— 1080 (ATCC CCL— 121)、 HeLa (ATCC CCL— 2)、 293 (ECACC 85120602) , Namalwa (ATCC CRL— 1432)、 Namalwa KJM— 1 (Cytotechnology, 1, 151 (1988)、 NM— F9細胞（DSM ACC2605、 WOO 5Z17130)および PER. C6細胞（ECACC No. 96022940、 US6855544)、サ ル細胞株である VERO (ATCC CCL— 1651)および COS— 7 (ATCC CRL— 1 651)、マウス細胞株である C127I (ATCC CRL— 1616)、 Sp2Z〇— Agl4 (AT CC CRL— 1581)、 NIH3T3 (ATCC CRL— 1658)、 NS0 (ATCC CRL— 18 27)、ラット細胞株である Y3 Agl. 2. 3. (ATCC CRL 1631)、 YO (ECACC No : 85110501)および YB2Z0 (ATCC CRL— 1662)、ハムスター細胞株であ る CHO— Kl (ATCC CCL— 61)、 CHO/dhfr" (ATCC CRL— 9096)、 CH 0/DG44[Proc. Natl. Acad. Sci. USA, 77, 4216 (1980) ]および BHK21 ( ATCC CRL— 10)、ィヌ細胞である MDCK( ATCC CCL— 34)などがあげられ る。鳥類に属する動物細胞としては、例えば-ヮトリ細胞株 SL— 29 (ATCC CRL — 29)など、魚類に属する動物細胞としては、例えばゼブラフィッシュ細胞株 ZF4 (A TCC CRL— 2050)など、昆虫類に属する動物細胞としては、例えば蛾（SOodoOt era frueiperda)細朐株 Sf 9 (ATCC CRL— 1711)などがそれぞれあげられる。 また、本発明に用いられる動物細胞として、ワクチン製造に使用される初代培養細胞 である、初代サル腎細胞、初代ゥサギ腎細胞、初代ニヮトリ胎児細胞、初代ゥズラ胎 児細胞などを用いてもよい。 [0051] Examples of animal cells belonging to mammals include the human cell line HL-60 (ATCC C CL—240), HT—1080 (ATCC CCL—121), HeLa (ATCC CCL—2), 293 (ECACC 85120602), Namalwa (ATCC CRL—1432), Namalwa KJM—1 (Cytotechnology, 1, 151 (1988) , NM—F9 cells (DSM ACC2605, WOO 5Z17130) and PER. C6 cells (ECACC No. 96022940, US6855544), Sal cell lines VERO (ATCC CCL— 1651) and COS—7 (ATCC CRL— 1 651) , Mouse cell lines C127I (ATCC CRL— 1616), Sp2Z〇—Agl4 (AT CC CRL—1581), NIH3T3 (ATCC CRL—1658), NS0 (ATCC CRL—18 27), rat cell line Y3 Agl 2. 3. (ATCC CRL 1631), YO (ECACC No: 85110501) and YB2Z0 (ATCC CRL—1662), hamster cell line CHO—Kl (ATCC CCL—61), CHO / dhfr ”(ATCC CRL — 9096), CH 0 / DG44 [Proc. Natl. Acad. Sci. USA, 77, 4216 (1980)] and BHK21 (ATCC CRL—10), Inu cell MDCK (ATCC CCL—34) Examples of animal cells belonging to avian species include the -bird cell line SL-29 (ATCC CRL-29). Examples of animal cells belonging to fish include zebrafish cell line ZF4 (A TCC CRL-2050). Examples of animal cells belonging to insects include moth (SOodoOt era frueiperda) cell line Sf 9 (ATCC CRL— In addition, as animal cells used in the present invention, primary monkey kidney cells, primary rabbit rabbit cells, primary chicken embryo cells, primary quail embryo cells, which are primary culture cells used for vaccine production. Cells or the like may be used.
[0052] ミエローマ細胞またはミエローマ細胞に由来する細胞としては、 Sp2Z0— Agl4、 NS0、Y3 Agl. 2. 3.、 YOまたは YB2Z0などがあげられる。卵巣細胞または卵 巣細胞に由来する細胞としては、 CHO—Kl、 CHOZdhfr—または CHOZDG44 などがあげられる。また、腎臓細胞としては、 293、 VERO、 COS— 7、 BHK21また は MDCKなどが、血球細胞としては HL— 60、 Namalwa, Namalwa KJM—lま たは NM— F9など力 子宫細胞としては HeLaなど力 結合組織細胞としては HT— 1080または NIH3T3など力 乳腺細胞としては C1271Iなど力 胚性網膜芽細胞と しては PER. C6などが、それぞれあげられる。  [0052] Examples of myeloma cells or cells derived from myeloma cells include Sp2Z0—Agl4, NS0, Y3 Agl. 2. 3., YO, and YB2Z0. Examples of cells derived from ovary cells or oocyte cells include CHO-Kl, CHOZdhfr-, or CHOZDG44. Kidney cells include 293, VERO, COS-7, BHK21, or MDCK, blood cells include HL-60, Namalwa, Namalwa KJM-l, or NM-F9. Examples of force connective tissue cells include HT-1080 and NIH3T3. Examples of force mammary cells include C1271I. Examples of force embryonic retinoblasts include PER. C6.
[0053] 本発明に用いられる動物細胞としては、物質を産生する動物細胞、変異処理を施 して物質を産生するようになった細胞、物質の生産に関与する遺伝子を含有する糸且 換え体ベクターで形質転換された細胞、 B細胞などの抗体産生細胞とミエローマ細 胞との融合細胞であるハイプリドーマなどが用いられる。また、本発明の細胞に物質 の発現量を上昇させるような変異処理を施した動物細胞を用いてもよい。 [0053] Animal cells used in the present invention include animal cells that produce a substance, and a mutation treatment. Cells that have produced substances, cells transformed with thread-recombinant vectors that contain genes involved in substance production, fusion cells of antibody-producing cells such as B cells and myeloma cells A certain high-pridoma is used. Animal cells obtained by subjecting the cells of the present invention to a mutation treatment that increases the expression level of the substance may also be used.
[0054] 変異処理を施して物質を産生するようになった細胞としては、所望の物質を生産で きるようにするために蛋白質の修飾酵素などに変異が導入された細胞などがあげら れ、例えば、所望の物質が糖蛋白質である場合には、糖鎖の構造を変化させるため に、種々の糖鎖修飾酵素に変異が導入された細胞などが用いられてもよい。 [0054] 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. For example, when 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.
物質の生産に関与する遺伝子を含有する組換え体ベクターで形質転換された細 胞としては、物質の生産に関与する DNAとプロモーターとを含む組換え体ベクター を、上記の本発明に用いられる動物細胞に導入することによって得られる細胞などが あげられる。  As a cell transformed with a recombinant vector containing a gene involved in the production of a substance, a recombinant vector containing a DNA involved in the production of the substance and a promoter is used as the animal used in the present invention. Examples thereof include cells obtained by introduction into cells.
[0055] 物質の生産に関与する DNAとしては、例えば、ペプチドなどの物質をコードする D NA、物質の生合成に関わる酵素または蛋白質をコードする DNAなどのいずれも用 いることがでさる。  [0055] As the 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 biosynthesis of the substance, and the like can be used.
本発明の方法により製造される物質としては、動物細胞が生産できる物質であれば いずれでもよい、例えば、ペプチド、リボザィムなどの生体触媒分子、ケラチン、コラ 一ゲン、エラスチン、レシリンおよびフイブ口インなどの細胞構造の形成'保持分子、 痘瘡ワクチン、ポリオワクチン、麻疹ワクチン、風疹ワクチン、おたふく風邪ワクチン、 狂犬病ワクチン、水痘ワクチン、ゥシ流行熱ワクチン、イバラキ病ワクチンおよびゥシ 伝染性気管炎ワクチンなどのワクチン、並びにアデノウイルスなどのウィルスなどがあ げられる。  The substance produced by the method of the present invention may be any substance that can be produced by animal cells, for example, biocatalytic molecules such as peptides and ribozymes, keratin, collagen, elastin, resilin, and five mouthin. `` Formation of the cell structure of '', such as pressure ulcer vaccine, polio vaccine, measles vaccine, rubella vaccine, mumps vaccine, rabies vaccine, varicella vaccine, ushi epidemic fever vaccine, Ibaraki disease vaccine and ushi infectious tracheitis vaccine Examples include vaccines and viruses such as adenovirus.
[0056] 本発明の方法により製造されるペプチドとしては、真核細胞由来のペプチドがあげ られ、好ましくは哺乳動物細胞由来のペプチドがあげられる。また、本発明において ペプチドとしては、所望のペプチドそのもの、当該ペプチドを含むペプチドの他、他 のペプチドと融合させた融合ペプチドであってもよ 、し、その部分断片であってもよ い。  [0056] Peptides produced by the method of the present invention include peptides derived from eukaryotic cells, preferably peptides derived from mammalian cells. In the present invention, the peptide may be a desired peptide itself, a peptide containing the peptide, a fusion peptide fused with another peptide, or a partial fragment thereof.
本発明の方法により製造されるペプチドの具体例としては、糖蛋白質または生理活 性を有するペプチドなどがあげられる。 Specific examples of the peptide produced by the method of the present invention include glycoprotein or physiological activity. And a peptide having sex.
[0057] 糖蛋白質としては、例えば、抗体、エリスロポイエチン (EPO) [J. Biol. Chem. , 2 52, 5558 ( 1977) ]、卜ロンボポイエチン (TPO) [Nature, 369 533 ( 1994) ]組織 型プラスミノーゲンァクチベータ、プロゥロキナーゼ、トロンボモジュリン、アンチトロン ビン III、プロテインお血液凝固因子 VII、血液凝固因子 VIII、血液凝固因子 IX、血 液凝固因子 X、血液凝固因子 XI、血液凝固因子 XII、プロトロンビン複合体、フイブリ ノゲン、アルブミン、性腺刺激ホルモン、甲状腺刺激ホルモン、上皮増殖因子 (EGF )、肝細胞増殖因子 (HGF)、ケラチノサイト増殖因子、ァクチビン、骨形成因子、顆 粒球コロニー刺激因子（G— CSF) ϋ. Biol. Chem. , 258, 9017 ( 1983) ]、マクロ ファージコ口-一刺激因子（Μ— CSF) ϋ. Exp. Med. , 173, 269 ( 1992) ]などの 幹細胞因子（SCF)、顆粒球—マクロファージコロニー刺激因子（GM— CSF) [J. Bi ol. Chem. , 252, 1998 ( 1977) ]、顆粒球—マクロファージコロニー刺激因子（G M - CSF) [J. Biol. Chem. , 252 1998 ( 1977) ]、インターフェロン α、インター フエロン j8、インターフェロン γ、インターロイキン一 2 (IL— 2) [Science, 193, 100 7 ( 1976) ]、インターロイキン 6、インターロイキン 10、インターロイキン 11、インター口 ィキン— 12 (IL— 12) ϋ. Leuc. Biol. , 55, 280 ( 1994) ]、可溶性インターロイキン 4受容体、腫瘍壊死因子 at、 Dnasel、ガラクトシダーゼ、 αダルコシダーゼ、ダルコ セレブロシダーゼ、ヘモグロビン、トランスフェリンおよびこれらの糖蛋白質の部分断 片などがあげられる。 [0057] Examples of glycoproteins include antibodies, erythropoietin (EPO) [J. Biol. Chem., 252, 5558 (1977)], 卜 ombopoietin (TPO) [Nature, 369 533 (1994)] tissue Plasminogen activator, prolokinase, thrombomodulin, antithrombin III, 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 morphogenetic factor, condylar granulocyte colony stimulating factor (G — CSF) ϋ. Biol. Chem., 258, 9017 (1983)], macrophage-mouth stimulating factor (Μ—CSF) ϋ. Exp. Med., 173, 269 (1992)] and other stem cell factors (SCF ), Granulocytes-macro Colony stimulating factor (GM-CSF) [J. Biol. Chem., 252, 1998 (1977)], granulocyte-macrophage colony stimulating factor (GM-CSF) [J. Biol. Chem., 252 1998 (1977) ], Interferon α, Interferon j8, Interferon γ, Interleukin 1 (IL-2) [Science, 193, 100 7 (1976)], Interleukin 6, Interleukin 10, Interleukin 11, Interoral Kinkin— Leuc. Biol., 55, 280 (1994)], soluble interleukin 4 receptor, tumor necrosis factor at, Dnasel, galactosidase, α-darcosidase, darcocerebrosidase, hemoglobin, transferrin and their For example, partial fragments of glycoproteins.
[0058] 抗体としては、 V、かなる抗原結合性を有する抗体でもよぐ例えば腫瘍関連抗原に 結合する抗体、アレルギーあるいは炎症に関連する抗原に結合する抗体、循環器疾 患に関連する抗原に結合する抗体、自己免疫疾患に関連する抗原に結合する抗体 、またはウィルスある!/ヽは細菌感染に関連する抗原に結合する抗体などがあげられる 。抗体のクラスはいずれでもよいが、 IgGクラスが好ましい。  [0058] Examples of antibodies include V, antibodies having such antigen binding properties, such as antibodies that bind to tumor-related antigens, antibodies that bind to antigens related to allergy or inflammation, and antigens related to cardiovascular diseases. Examples include antibodies that bind, antibodies that bind to antigens associated with autoimmune diseases, or viruses! /! That bind to antigens associated with bacterial infections. The antibody class may be any, but the IgG class is preferred.
[0059] 本発明の方法により製造される抗体としては、抗体の一部分を含む断片などが包 含され、例えば、 Fab (Fragment of antigen binding の略）、 Fab，、 F (ab，）  [0059] The antibody produced by the method of the present invention includes a fragment containing a part of the antibody. For example, Fab (abbreviation of Fragment of antigen binding), Fab, F (ab,)
2 2
、一本鎖抗体（single chain Fv ;以下、 scFvと称す）およびジスルフイド安定化抗 体（disulfide stabilized Fv ;以下、 dsFvと称す）や、抗体の Fc領域を含む融合蛋 白質などがあげられる。 Single-chain antibodies (single chain Fv; hereinafter referred to as scFv) and disulfide stabilized antibodies (hereinafter referred to as dsFv) and fusion proteins containing the Fc region of the antibody. Examples include white matter.
[0060] 抗体としては、動物に抗原を免疫し、被免疫動物の脾臓細胞などの抗体産生細胞 より作製したハイプリドーマ細胞が分泌する抗体の他、遺伝子組換え技術により作製 された抗体、すなわち、抗体遺伝子を挿入した抗体発現ベクターを、宿主細胞へ導 入することにより取得された抗体などがあげられる。抗体としては、具体的には、ハイ プリドーマが生産する抗体、ヒト型キメラ化抗体、ヒトイ匕抗体またはヒト抗体などをあげ ることがでさる。  [0060] Examples of antibodies include antibodies produced by gene recombination techniques, in addition to antibodies secreted by high-pridoma cells produced from antibody-producing cells such as spleen cells of immunized animals after immunization of the animal with antigens, Examples thereof include antibodies obtained by introducing an antibody expression vector into which an antibody gene has been inserted into a host cell. Specific examples of the antibody include an antibody produced by Hypridoma, a human chimerized antibody, a human rabbit antibody, a human antibody, and the like.
[0061] ヒト型キメラ抗体は、ヒト以外の動物の抗体重鎖可変領域 (以下、重鎖は H鎖として 、可変領域は V領域として HVまたは VHとも ヽぅ）および抗体軽鎖可変領域 (以下、 軽鎖は L鎖として LVまたは VLともいう）とヒト抗体の重鎖定常領域 (以下、定常領域 は C領域として CHとも 、う）およびヒト抗体の軽鎖定常領域 (以下、 CLとも 、う）とから なる抗体をいう。ヒト以外の動物としては、マウス、ラット、ノヽムスター、ラビットなど、ノヽ イブリドーマを作製することが可能であれば、いかなるものも用いることができる。  [0061] A human chimeric antibody is a non-human animal antibody heavy chain variable region (hereinafter referred to as heavy chain as H chain, variable region as V region as HV or VH) and antibody light chain variable region (hereinafter referred to as "H chain"). The light chain is also referred to as LV or VL as the L chain) and the heavy chain constant region of human antibodies (hereinafter, the constant region is also referred to as CH as C region) and the light chain constant region of human antibodies (hereinafter also referred to as CL). ). Any animal other than a human can be used as long as it can produce a hybridoma, such as a mouse, rat, nomstar, or rabbit.
[0062] ヒト型キメラ抗体は、モノクローナル抗体を生産するハイブリドーマより VHおよび VL をコードする cDNAを取得し、ヒト抗体 CHおよびヒト抗体 CLをコードする遺伝子を有 する宿主細胞用発現ベクターにそれぞれ挿入してヒト型キメラ抗体発現ベクターを構 築し、宿主細胞へ導入することにより発現させ、製造することができる。  [0062] For human chimeric antibodies, 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. Thus, a human chimeric antibody expression vector can be constructed and introduced into a host cell for expression and production.
ヒト型キメラ抗体の CHとしては、ヒトイムノグロブリン (以下、 hlgという）に属すればい かなるものでもよいが、 hlgGクラスのものが好適であり、さらに hlgGクラスに属する hi gGl、 hIgG2、 hIgG3、 hIgG4といったサブクラスのいずれも用いることができる。ま た、ヒト型キメラ抗体の CLとしては、 hlgに属すればいかなるものでもよぐ κクラスあ るいはえクラスのものを用いることができる。  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 hlgG class, and further higGl, hIgG2, hIgG3, Any of the subclasses such as hIgG4 can be used. In addition, as the CL of the human chimeric antibody, any of those belonging to hlg can be used, and those of κ class or fly class can be used.
[0063] ヒト化抗体としては、ヒト以外の動物の抗体の VHおよび VLのヒト型相同性決定領 域 (complementarity determining region :以下、 CDRと ヽつ)のアミノ酸目 ti歹 Uを ヒト抗体の VHおよび VLの適切な位置に移植して作製されたヒト型 CDR移植抗体な どがあげられる。  [0063] As a humanized antibody, VH and VL of a non-human animal antibody, amino acid ti 歹 U of human type homology determining region (hereinafter referred to as CDR) is designated as VH of human antibody. And human-type CDR-grafted antibody produced by transplanting at an appropriate position of VL.
ヒト型 CDR移植抗体は、ヒト以外の動物の抗体の VHおよび VLの CDR配列を任意 のヒト抗体の VHおよび VLの CDR配列に移植した V領域をコードする cDNAを構築 し、ヒト抗体の CHおよびヒト抗体の CLをコードする遺伝子を有する宿主細胞用発現 ベクターにそれぞれ挿入してヒト型 CDR移植抗体発現ベクターを構築し、該発現べ クタ一を宿主細胞へ導入することによりヒト型 CDR移植抗体を発現させ、製造するこ とがでさる。 Human-type CDR-grafted antibody is a cDNA encoding the V region in which the VH and VL CDR sequences of non-human animal antibodies are grafted to the VH and VL CDR sequences of any human antibody. And constructing a human CDR-grafted antibody expression vector by inserting it into a host cell expression vector having genes encoding human antibody CH and human antibody CL, and introducing the expression vector into the host cell. To express and produce human CDR-grafted antibodies.
[0064] ヒト型 CDR移植抗体の CHとしては、 hlgに属すればいかなるものでもよいが、 hlgG クラスのものが好適であり、さらに hlgGクラスに属する hlgG 1、 hIgG2、 hIgG3、 hlg G4といったサブクラスのいずれも用いることができる。また、ヒト型 CDR移植抗体の C Lとしては、 hlgに属すればいかなるものでもよぐ κクラスまたは λクラスのものを用 いることがでさる。  [0064] As the CH of the human CDR-grafted antibody, any CH can be used as long as it belongs to hlg, but the hlgG class is preferable, and hlgG 1, hIgG2, hIgG3, and hlg G4 belonging to the hlgG class are preferred. Either can be used. As the CL of the human CDR-grafted antibody, any κ class or λ class antibody belonging to hlg can be used.
[0065] 本発明の方法により製造される抗体の具体例としては、腫瘍関連抗原に結合する 抗体としては、抗 GD2抗体 [Anticancer Res. , 13, 331 (1993)]、抗 GD3抗体 [ Cancer Immunol. Immunother. , 36, 260(1993)]、抗 GM2抗体 [Cancer Res. , 54, 1511(1994)]、抗 HER2抗体 [Proc. Natl. Acad. Sci. USA, 89, 4 285(1992)]、抗 CD52抗体 [Nature, 332, 323(1988)]、抗 MAGE抗体（Briti sh J. Cancer, 83, 493(2000))、抗 HMl. 24抗体 [Molecular Immunol. , 3 6, 387(1999)]、抗副甲状腺ホルモン関連蛋白 PTHrP抗体 [Cancer, 88, 2909 (2000)]、抗 FGF8抗体 [Proc. Natl. Acad. Sci. USA, 86, 9911(1989)]抗 塩基性繊維芽細胞増殖因子抗体、抗 FGF8受容体抗体 Q[. Biol. Chem. , 265, 1 6455(1990)]、抗塩基性繊維芽細胞増殖因子受容体抗体、抗インスリン様増殖因 子抗体、抗インスリン様増殖因子受容体抗体 Q[. Neurosci. Res. , 40, 647(1995 )]、抗 PMSA抗体 Q[. Urology, 160, 2396(1998)]、抗血管内皮細胞増殖因子 抗体 [Cancer Res. , 57, 4593(1997)]、抗血管内皮細胞増殖因子受容体抗体 [Oncogene, 19, 2138(2000)]、抗 CA125抗体、抗 17— 1A抗体、抗インテグリ ン a vj83抗体、抗 CD33抗体、抗 CD22抗体、抗 HLA抗体、抗 HLA— DR抗体、 抗 CD20抗体、抗 CD 19抗体、抗 EGF受容体抗体 [Immunology Today、 21, 4 03(2000)]、抗 CD10抗体 [American Journal of Clinical Pathology, 113 , 374 (2000)]などがあげられる。  [0065] Specific examples of antibodies produced by the method of the present invention include anti-GD2 antibodies [Anticancer Res., 13, 331 (1993)], anti-GD3 antibodies [Cancer Immunol] Immunother., 36, 260 (1993)], anti-GM2 antibody [Cancer Res., 54, 1511 (1994)], anti-HER2 antibody [Proc. Natl. Acad. Sci. USA, 89, 4 285 (1992)] , Anti-CD52 antibody [Nature, 332, 323 (1988)], anti-MAGE antibody (British J. Cancer, 83, 493 (2000)), anti-HMl. 24 antibody [Molecular Immunol., 3, 6, 387 (1999) ], Anti-parathyroid hormone related protein PTHrP antibody [Cancer, 88, 2909 (2000)], anti-FGF8 antibody [Proc. Natl. Acad. Sci. USA, 86, 9911 (1989)] anti-basic fibroblast growth factor Antibody, anti-FGF8 receptor antibody Q [. Biol. Chem., 265, 1 6455 (1990)], anti-basic fibroblast growth factor receptor antibody, anti-insulin-like growth factor antibody, anti-insulin-like growth factor receptor Antibody Q [. Neurosci. Res., 40, 647 (1995)], anti-PMSA antibody Q [. Urology, 160, 2396 (1998)] Anti-vascular endothelial growth factor antibody [Cancer Res., 57, 4593 (1997)], anti-vascular endothelial growth factor receptor antibody [Oncogene, 19, 2138 (2000)], anti-CA125 antibody, anti-17-1A antibody, Anti-integrin a vj83 antibody, anti-CD33 antibody, anti-CD22 antibody, anti-HLA antibody, anti-HLA-DR antibody, anti-CD20 antibody, anti-CD 19 antibody, anti-EGF receptor antibody [Immunology Today, 21, Apr 03 (2000) And anti-CD10 antibody [American Journal of Clinical Pathology, 113, 374 (2000)].
[0066] アレルギーあるいは炎症に関連する抗原に結合する抗体の具体例としては、抗イン ターロイキン 6抗体 [Immunol. Rev. , 127, 5(1992)]、抗インターロイキン 6受容 体抗体 [Molecular Immunol. , 31, 371 (1994)]、抗インターロイキン 5抗体 [I mmunol. Rev. , 127, 5(1992)]、抗インターロイキン 5受容体抗体、抗インター口 ィキン 4抗体 [Cytokine, 3, 562(1991)]、抗インターロイキン 4受容体抗体 ϋ. Im munol. Meth. , 217, 41 (1998)]、抗腫瘍壊死因子抗体 [Hybridoma, 13, 18 3(1994)]、抗腫瘍壊死因子受容体抗体 [Molecular Pharmacol. , 58, 237(2 000)]、抗 CCR4抗体 [Nature, 400, 776(1999)]、抗ケモカイン抗体 ϋ. Immu nol. Meth. , 174, 249(1994)]、抗ケモカイン受容体抗体 ϋ. Exp. Med. , 186 , 1373(1997)]、抗 IgE抗体、抗 CD23抗体、抗 CDl la抗体 [Immunology Tod ay, 21, 403(2000)]、抗 CRTH2抗体 ϋ. Immunol. , 162, 1278(1999)]、抗 CCR8抗体 (W099Z25734)、抗 CCR3抗体（US6207155)などがあげられる。 [0066] Specific examples of antibodies that bind to antigens associated with allergies or inflammation include anti-ins Turleukin 6 antibody [Immunol. Rev., 127, 5 (1992)], anti-interleukin 6 receptor antibody [Molecular Immunol., 31, 371 (1994)], anti-interleukin 5 antibody [I mmunol. Rev., 127 , 5 (1992)], anti-interleukin 5 receptor antibody, anti-interleukin 4 antibody [Cytokine, 3, 562 (1991)], anti-interleukin 4 receptor antibody ϋ. Im munol. Meth., 217, 41 (1998)], anti-tumor necrosis factor antibody [Hybridoma, 13, 18 3 (1994)], anti-tumor necrosis factor receptor antibody [Molecular Pharmacol., 58, 237 (2 000)], anti-CCR4 antibody [Nature, 400 , 776 (1999)], anti-chemokine antibody ϋ. Immu nol. Meth., 174, 249 (1994)], anti-chemokine receptor antibody ϋ. Exp. Med., 186, 1373 (1997)], anti-IgE antibody, Anti-CD23 antibody, anti-CDl la antibody [Immunology Tod ay, 21, 403 (2000)], anti-CRTH2 antibody Immuno. Immunol., 162, 1278 (1999)], anti-CCR8 antibody (W099Z25734), anti-CCR3 antibody (US6207155) Etc.
[0067] 循環器疾患に関連する抗原に結合する抗体の具体例としては、抗 GpIIbZlIIa抗 体 Ci. Immunol. , 152, 2968(1994)]、抗血小板由来増殖因子抗体 [Science, 253. 1129(1991)]、抗血小板由来増殖因子受容体抗体 Q[. Biol. Chem. , 272 , 17400 (1997)]または抗血液凝固因子抗体 [Circulation, 101, 1158(2000)] などが挙げられる。 [0067] Specific examples of antibodies that bind to an antigen associated with cardiovascular disease include anti-GpIIbZlIIa antibody Ci. Immunol., 152, 2968 (1994)], anti-platelet-derived growth factor antibody [Science, 253.1129 ( 1991)], antiplatelet-derived growth factor receptor antibody Q [. Biol. Chem., 272, 17400 (1997)] or anti-blood coagulation factor antibody [Circulation, 101, 1158 (2000)].
[0068] 自己免疫疾患に関連する抗原に結合する抗体の具体例としては、抗自己 DNA抗 体 [Immunol. Letters, 72, 61 (2000)]、抗 CDl la抗体、抗 ICAM3抗体、抗 C D80抗体、抗 CD2抗体、抗 CD3抗体、抗 CD4抗体、抗インテグリン α 4 j87抗体、 抗 CD40L抗体、抗 IL 2受容体抗体 [Immunology Today, 21, 403(2000)] などがあげられる。  [0068] Specific examples of antibodies that bind to antigens associated with autoimmune diseases include anti-self DNA antibodies [Immunol. Letters, 72, 61 (2000)], anti-CDlla antibodies, anti-ICAM3 antibodies, anti-CD80. Antibody, anti-CD2 antibody, anti-CD3 antibody, anti-CD4 antibody, anti-integrin α4 j87 antibody, anti-CD40L antibody, anti-IL 2 receptor antibody [Immunology Today, 21, 403 (2000)].
[0069] ウィルスあるいは細菌感染に関連する抗原に結合する抗体の具体例としては、抗 g pi 20抗体 [Structure, 8, 385(2000)]、抗 CD4抗体 ϋ. Rheumatology, 25, 2 065(1998)]、抗 CCR4抗体、抗ベロ毒素抗体 Q[. Clin. Microbiol. , 37, 396 ( 1999)]などがあげられる。  [0069] Specific examples of antibodies that bind to an antigen associated with a virus or bacterial infection include anti-gpi 20 antibody [Structure, 8, 385 (2000)], anti-CD4 antibody ϋ. Rheumatology, 25, 2 065 (1998). )], Anti-CCR4 antibody, anti-verotoxin antibody Q [. Clin. Microbiol., 37, 396 (1999)].
[0070] 生理活性を有するペプチドとしては、とくに制限はな 、が、例えば上記の糖蛋白質 の部分断片のうち、該糖蛋白質の活性を維持しているペプチドなどがあげられる。ま た、糖蛋白質が酵素である場合には、酵素活性を調節するペプチドまたは酵素の構 造を保持するペプチドなども包含される。酵素の活性を調節するペプチドとしては、 例えば、糖蛋白質のァゴニストまたはアンタゴニストとして機能するペプチドなどがあ げられる。ァゴニストとしては、糖蛋白質の活性を亢進する活性を有するペプチドなど があげられ、具体的には、ソマトスタチン誘導体、ソマトロビン、心房性ナトリウム利尿 ペプチド、グルカゴン、インスリン、インスリン様成長因子、性腺刺激ホルモン放出ホ ルモンなどがあげられる。アンタゴ-ストとしては、糖蛋白質の活性を抑制する活性を 有するペプチドなどがあげられ、具体的には、ぺグピソマントなどがあげられる。 [0070] The peptide having physiological activity is not particularly limited, and examples thereof include peptides that maintain the activity of the glycoprotein among the partial fragments of the glycoprotein. In addition, when the glycoprotein is an enzyme, a peptide or enzyme structure that regulates the enzyme activity. Peptides that retain the structure are also included. Examples of peptides that regulate the activity of the enzyme include peptides that function as glycoprotein agonists or antagonists. Examples of agonists include peptides having the activity of enhancing glycoprotein activity, and specifically include somatostatin derivatives, somatrobin, atrial natriuretic peptide, glucagon, insulin, insulin-like growth factor, gonadotropin-releasing hormone. Lemon. Examples of the antagonist include peptides having an activity of suppressing the activity of glycoprotein, and specifically, pegpisomanto.
[0071] 本発明の方法によりペプチドを製造する場合に用いられる動物細胞としては、当該 ペプチドを製造できれば 、ずれの動物細胞を用いてもょ 、が、製造するペプチドをコ ードする遺伝子を含有するベクターが導入された形質転換体が、好ましく用いられる ペプチドをコードする遺伝子を含有するベクターが導入された形質転換された細胞 は、例えばペプチドをコードする DNAとプロモーターを含む糸且換え体ベクターを、宿 主細胞に導入することによって得られる。宿主細胞としては上記の動物細胞が用いら れる。  [0071] The animal cell used in producing the peptide by the method of the present invention contains a gene that codes the peptide to be produced, although the animal cell may be used as long as the peptide can be produced. For example, a transformed cell into which a vector containing a gene encoding a peptide that is preferably used is introduced can be obtained by using, for example, a thread-recombinant vector containing a DNA encoding a peptide and a promoter. , Obtained by introducing into the host cell. The animal cells described above are used as host cells.
[0072] ペプチドをコードする遺伝子を含有するベクターを調製するために用いられるベタ ターとしては、例えば、 pcDNAI、 pcDM8 (フナコシ社製）、 pAGE107 [特開平 3— 22979号、 Cytotechnology, 3, 133 (1990) ]、 pAS3— 3 (特開平 2— 227075 号）、 pcDM8 [Nature, 329. 840 (1987) ]、 pcDNAlZAmp (Invitrogen社製） 、 pREP4 (Invitrogen社製）、 pAGE103 Q[. Biochem. , 101, 1307 (1987) ]、 ρ AGE210などがあげられる。  [0072] Examples of the vector used for preparing a vector containing a gene encoding a peptide include pcDNAI, pcDM8 (manufactured by Funakoshi), pAGE107 [Japanese Patent Laid-Open No. 3-22979, Cytotechnology, 3, 133 ( 1990)], pAS3-3 (JP-A-2-227075), pcDM8 [Nature, 329. 840 (1987)], pcDNAlZAmp (Invitrogen), pREP4 (Invitrogen), pAGE103 Q [. Biochem., 101 , 1307 (1987)], and ρ AGE210.
[0073] プロモーターとしては、本発明で用いる動物細胞中で機能するものであればいず れも用いることができ、例えば、サイトメガロウィルス (CMV)の IE (immediate early )遺伝子のプロモーター、 SV40の初期プロモーター、レトロゥイノレスのプロモーター 、メタ口チォネインプロモーター、ヒートショックプロモーター、 SR aプロモーターなど があげられる。また、ヒト CMVの IE遺伝子のェンハンサーなどをプロモーターとともに 用いてもよい。  [0073] Any promoter can be used as long as it functions in animal cells used in the present invention. For example, cytomegalovirus (CMV) IE (immediate early) gene promoter, early SV40 Examples include promoters, retrowinoles promoters, meta-mouthone promoters, heat shock promoters, SRa promoters, and the like. In addition, an enhancer of the IE gene of human CMV may be used together with a promoter.
[0074] 宿主細胞への組換え体ベクターの導入方法としては、当該細胞に DNAを導入す る方法であればいずれも用いることができ、例えば、エレクト口ポレーシヨン法 [Cytot echnology, 3, 133 (1990) ]、リン酸カルシウム法（特開平 2— 227075)、リポフエ クシヨン法 [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)、 Virology, 52, 4 56 (1973) ]などがあげられる。 [0074] As a method for introducing a recombinant vector into a host cell, DNA is introduced into the cell. Any method can be used, for example, the electopore position method [Cytot echnology, 3, 133 (1990)], the calcium phosphate method (JP-A-2-227075), the lipofusion method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987), Virology, 52, 4 56 (1973)].
[0075] 本発明に用いられる形質転換細胞としては、具体的には、抗 GD3ヒト型キメラ抗体 を生産する形質転換細胞 7— 9 51 (FERM BP— 6691)、抗 CCR4キメラ抗体を 生産する形質転換細胞 KM2760 (FERM BP 7054)、抗 CCR4ヒト化抗体を生 産する开質転換細胞 KM8759 (FERM BP— 8129)、 KM8760 (FERM BP— 8130)、 709 LCA—500D株 (FERM BP— 8239)、抗 IL 5受容体 α鎖キメラ抗 体を生産する形質転換細胞 ΚΜ7399 (FERM BP— 5649)、抗 IL 5受容体 α鎖 ヒト型 CDR移植抗体を生産する形質転換細胞 ΚΜ8399 (FERM BP— 5648)およ び KM9399 (FERM BP— 5647)、抗 GM2ヒト型 CDR移植抗体を生産する形質 転換細胞 KM8966 (FERM BP— 5105)、 KM8967 (FERM BP— 5106)、 K M8969 (FERM BP— 5527)、 KM8970 (FERM BP— 5528)、抗 CD20抗体 を生産する形質転換株 Ms704— CD20 (FERM BP— 10092)および ΑΤΠΙを生 産する开質転換細胞 Ms705-pKAN— ΑΤΠΙ (FERM BP— 8472)などがあげら れる。 [0075] Specific examples of the 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), transformant cells producing anti-CCR4 humanized antibodies KM8759 (FERM BP—8129), KM8760 (FERM BP—8130), 709 LCA-500D strain (FERM BP—8239), Transformed cells producing anti-IL 5 receptor α-chain chimeric antibody ΚΜ7399 (FERM BP—5649), transformed cells producing anti-IL 5 receptor α-chain human CDR-grafted antibody 998399 (FERM BP—5648) And KM9399 (FERM BP—5647), transformed cells producing anti-GM2 human CDR-grafted antibody KM8966 (FERM BP—5105), KM8967 (FERM BP—5106), K M8969 (FERM BP—5527), KM8970 ( FERM BP—5528), transformant Ms704—CD20 (FERM BP—10092) that produces anti-CD20 antibodies, and transformant Ms705-pKAN—ΑΤΠΙ (FE RM BP— 8472).
[0076] 本発明にお 、て動物細胞を培養する方法としては、所望の物質を生産できる培養 方法であれば、バッチ培養、リピートバッチ培養、フヱドバッチ培養、パーフュージョン 培養など、いかなる培養方法を用いてもよいが、フヱドバッチ培養またはパーフュー ジョン培養が好ましく用いられる。  [0076] In the present invention, as a method for culturing animal cells, any culture method such as batch culture, repeat batch culture, food batch culture, and perfusion culture can be used as long as it can produce a desired substance. However, food batch culture or perfusion culture is preferably used.
フエドバツチ培養は、生理活性物質、栄養因子などを連続的、または間欠的に少量 ずつ追加供給する培養方法である。フエドバツチ培養は、細胞の代謝効率が高ぐ培 養液中の老廃物が蓄積されることによる培養細胞の到達細胞密度の低下を防止する ことができる。また、回収された培養液中の所望の物質は、ノ ツチ培養で得られた場 合に比べて高濃度であるため、該物質の分離'精製が容易で、ノ ツチ培養に比べ、 培地当たりの該物質の生産量を増大させることができる。さらには、培地に添加するリ ダナン類、フラボノイド類、ヒストン脱ァセチルイ匕酵素阻害剤、テルぺノイド類、コウジ 酸類力も選ばれる少なくとも 1つの物質の濃度の制御が、バッチ培養よりも容易であ る。 The Fuedbachi culture is a culture method in which physiologically active substances, nutrient factors, etc. are additionally supplied in small amounts continuously or intermittently. The Fuedbatch culture can prevent a decrease in the cell density of the cultured cells due to accumulation of waste products in a culture solution that increases the metabolic efficiency of the cells. In addition, since the desired substance in the collected culture medium has a higher concentration than that obtained in notch culture, the substance can be easily separated and purified, and compared to notch culture, The production amount of the substance can be increased. In addition, rydanans, flavonoids, histone deacetylase inhibitors, terpenoids, koji It is easier to control the concentration of at least one substance whose acid power is selected than batch culture.
[0077] パーフュージョン培養は、培養液と細胞とを分離する装置により効率的に分離され 、濃縮された細胞が元の培養槽に戻り、減少した分の新鮮培地が培養槽に新たに供 給される方法である。該方法は、培養槽内の培養環境が常に良好に保たれ、また、 新鮮培地を供給することにより槽内の浸透圧を制御することができるため、培地中の リグナン類、フラボノイド類、ヒストン脱ァセチルイ匕酵素阻害剤、テルぺノイド類、コウジ 酸類力 選ばれる少なくとも 1つの物質の濃度を制御するために好ましい方法である  [0077] Perfusion culture is efficiently separated by a device that separates the culture solution and cells, the concentrated cells are returned to the original culture tank, and the reduced amount of fresh medium is newly supplied to the culture tank. Is the method. In this method, the culture environment in the culture tank is always kept good, and the osmotic pressure in the tank can be controlled by supplying a fresh medium, so that lignans, flavonoids, and histones in the medium are removed. Acetylenic enzyme inhibitor, terpenoids, kojic acid power is a preferred method for controlling the concentration of at least one selected substance
[0078] 本発明にお 、て用いられる培養方法は、用いる動物細胞に適した方法であれば 、 ずれでもよいが、通常 pH6〜8、 30〜40°Cなどの条件下で 3〜20日間、パーフュー ジョン培養では 3〜60日間行う。また、培養中必要に応じて、ストレプトマイシン、ベニ シリンなどの抗生物質を培地に添加してもよい。なお、溶存酸素濃度制御、 pH制御 、温度制御、攪拌などは通常の動物細胞の培養に用いられる方法に準じて行うこと ができる。 [0078] The culture method used in the present invention may be any method as long as it is suitable for the animal cells to be used, but usually 3 to 20 days under conditions of pH 6 to 8, 30 to 40 ° C, etc. In perfusion culture, carry out for 3 to 60 days. In addition, antibiotics such as streptomycin and benicillin may be added to the medium as needed during culture. The dissolved oxygen concentration control, pH control, temperature control, stirring, etc. can be carried out according to the method used for normal animal cell culture.
[0079] 上記のようにして、リグナン類、フラボノイド類、ヒストン脱ァセチルイ匕酵素阻害剤、テ ルぺノイド類、コウジ酸類力 選ばれる少なくとも 1つの物質を含有する培地中で物 質を生産する能力を有する動物細胞を培養し、培養物中に所望の物質を生成蓄積 させ、該培養物から所望の物質を採取することにより、所望の物質を製造することが できる。  [0079] As described above, ability to produce substances in a medium containing at least one substance selected from lignans, flavonoids, histone deacetylase inhibitors, terpenoids, and kojic acid The desired substance can be produced by culturing animal cells having the above, producing and accumulating the desired substance in the culture, and collecting the desired substance from the culture.
所望の物質がペプチドである場合、本発明の製造方法としては、宿主細胞内にぺ プチドを生産させる直接発現方法、宿主細胞外にペプチドを分泌生産させる方法（ モレキュラー 'クロー-ング第 2版)などがあげられる。  When the desired substance is a peptide, the production method of the present invention includes a direct expression method in which the peptide is produced in the host cell, a method in which the peptide is secreted and produced outside the host cell (Molecular 'Cloning 2nd Edition). Etc.
[0080] ペプチドは、ポールソンらの方法 i. Biol. Chem. , 264, 17619 (1989)〕、ロウら の方法〔Proc. Natl. Acad. Sci. USA, 86, 8227 (1989)、 Genes Develop. , 4, 1288 (1990)〕、または特開平 5— 336963、 WO94Z23021などに記載の方法 を準用することにより、宿主細胞外へ積極的に分泌させることができる。すなわち、遺 伝子組換えの手法を用いて、所望のペプチドの N末端にシグナルペプチドを結合さ せた形で発現させることにより、ペプチドを宿主細胞外に積極的に分泌させることが できる。 [0080] Peptides can be prepared by the method of Paulson et al., I. Biol. Chem., 264, 17619 (1989)], the method of Law et al. [Proc. Natl. Acad. Sci. USA, 86, 8227 (1989), Genes Develop. , 4, 1288 (1990)], or by applying the method described in JP-A-5-336963, WO94Z23021 etc., it can be actively secreted outside the host cell. In other words, a signal peptide is bound to the N-terminus of the desired peptide using a gene recombination technique. By expressing in the form, the peptide can be actively secreted outside the host cell.
[0081] また、特開平 2— 227075に記載されている方法に準じて、ジヒドロ葉酸還元酵素 遺伝子などを用いた遺伝子増幅系を利用することにより、ペプチドの生産量を上昇さ せることちでさる。  [0081] Further, in accordance with the method described in JP-A-2-227075, by using a gene amplification system using a dihydrofolate reductase gene or the like, it is possible to increase the peptide production amount. .
本発明の方法により製造されるペプチドは、例えば、通常のペプチドの単離精製法 などを用いて単離精製することができる。  The peptide produced by the method of the present invention can be isolated and purified using, for example, a normal peptide isolation and purification method.
[0082] 本発明の方法により製造されるペプチドが細胞内に溶解状態で発現した場合には 、培養終了後、細胞を遠心分離により回収し、水系緩衝液にけん濁後、超音波破砕 機、フレンチプレス、マントンガウリンホモゲナイザー、ダイノミルなどにより細胞を破砕 し、無細胞抽出液を得る。該無細胞抽出液を遠心分離することにより得られる上清か ら、通常の酵素の単離精製法、即ち、溶媒抽出法、硫安などによる塩析法、脱塩法、 有機溶媒による沈殿法、ジェチルアミノエチル（DEAE)—セファロース、 DIAION HPA- 75 (三菱化成社製)などのレジンを用いた陰イオン交換クロマトグラフィー法 、 S—セファロース FF (フアルマシア社製）などのレジンを用いた陽イオン交換クロマト グラフィ一法、ブチルセファロース、フエ-ルセファロースなどのレジンを用いた疎水 性クロマトグラフィー法、分子篩を用いたゲルろ過法、プロテイン Aを用いたァフィ- ティークロマトグラフィー法、クロマトフォーカシング法と 、つた電点電気泳動などの電 気泳動法などを、単独あるいは組み合わせて用いることにより、粗精製標品または精 製標品を得ることができる。  [0082] When the peptide produced by the method of the present invention is expressed in a dissolved state in the cells, the cells are collected by centrifugation after culturing, suspended in an aqueous buffer, and then subjected to an ultrasonic crusher, Crush the cells with a French press, a Menton Gaurin homogenizer, or Dynomill to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, an ordinary enzyme isolation and purification method, that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, Anion-exchange chromatography using resin such as Jetylaminoethyl (DEAE) -Sepharose, DIAION HPA-75 (Mitsubishi Kasei), Cation using resin such as S-Sepharose FF (Falmasia) An exchange chromatography method, a hydrophobic chromatography method using a resin such as butyl sepharose, ferrule sepharose, a gel filtration method using a molecular sieve, a affinity chromatography method using protein A, a chromatofocusing method, By using an electrophoretic method such as Tatsuta Electrophoresis alone or in combination, a crudely purified sample or a purified sample Can be obtained.
[0083] 本発明の方法により製造されるペプチドが細胞外に分泌された場合には、培養上 清に該ペプチドを回収することができる。即ち、該培養物を上記と同様の遠心分離な どの手法により処理することにより培養上清を取得し、該培養上清から、上記と同様 の単離精製法を用いることにより、粗精製標品または精製標品を得ることができる。 また本発明の方法において、ペプチドの比生産速度（SPR: Specific productio n rate)を増大させることによって、ペプチドの生産性を向上させることができる。  [0083] When the peptide produced by the method of the present invention is secreted extracellularly, the peptide can be recovered in the culture supernatant. That is, a culture supernatant is obtained by treating the culture by a technique such as centrifugation as described above, and a crudely purified sample is obtained from the culture supernatant by using the same isolation and purification method as described above. Alternatively, a purified sample can be obtained. In the method of the present invention, the productivity of the peptide can be improved by increasing the specific product rate (SPR) of the peptide.
[0084] 物質の SPRは、物質の生産細胞あたりの生産される物質の量力 計算される。具 体的には、培養期間を通して生産された物質の量を、培養期間中に生存していた物 質の生産細胞数で除することにより、計算される。 [0084] The SPR of a substance is calculated by the amount of substance produced per substance producing cell. Specifically, the amount of substance produced throughout the culture period is the amount of material that survived during the culture period. Calculated by dividing by the number of quality cells produced.
本発明によれば、リグナン類、フラボノイド類、ヒストン脱ァセチルイ匕酵素阻害剤、テ ルぺノイド類、コウジ酸類カゝら選ばれる少なくとも 1つの物質を培地に添加すること〖こ より、物質の生合成または分泌の活性ィ匕などを通じて、動物細胞の細胞あたりの物質 の生産性を向上させることができる。  According to the present invention, by adding at least one substance selected from lignans, flavonoids, histone deacetylase inhibitors, terpenoids, and kojic acid to the medium, Through the activity of synthesis or secretion, the productivity of substances per animal cell can be improved.
[0085] 以下の実施例により本発明をより具体的に説明するが、実施例は本発明の単なる 例示にすぎず、本発明の範囲を限定するものではない。  [0085] The present invention will be described more specifically with reference to the following examples. However, the examples are merely illustrative of the present invention and do not limit the scope of the present invention.
実施例 1  Example 1
[0086] 抗体の無血清フ ドバツチ培養  [0086] Serum-free food culture of antibodies
WO03/046174の実施例 3に記載の抗 CCR4キメラ抗体を生産する 503LCA5 OOD株 (FERM BP— 8239)を用いて以下のフエドバッチ培養を行った。  Using the 503LCA5 OOD strain (FERM BP-8239) that produces the anti-CCR4 chimeric antibody described in Example 3 of WO03 / 046174, the following fed-batch culture was performed.
本培養までの拡大培養用培地には、 EX-CELL™ 302培地 (JRH社製）に、 Met hotrexate (以下、 MTXと記す） 500nmol/L (シグマアルドリッチ社製）、 L グルタ ミン（和光純薬社製） 1. 75gZLを添カ卩した培地を用いた。 125mL、 250mLまたは 1 OOOmL容量の三角フラスコ（コ一-ング社製）に約 10〜30%量の上記培地を入れ、 3 X 105細胞/ mLとなるよう〖こ 503 LCA— 500D株の細胞懸濁液を播種した。 35 °Cで 4日間培養し、本培養の播種に必要な細胞数が獲得されるまで複数回継代した The medium for expansion until the main culture is EX-CELL ™ 302 medium (manufactured by JRH), Met hotrexate (hereinafter referred to as MTX) 500 nmol / L (manufactured by Sigma-Aldrich), L glutamine (Wako Pure Chemicals) 1. A medium supplemented with 75 g ZL was used. Place approximately 10-30% of the above medium in a 125 mL, 250 mL or 1 OOOmL Erlenmeyer flask (manufactured by Corning Co., Ltd.) to obtain 3 X 10 5 cells / mL. 503 LCA- 500D strain cells The suspension was seeded. Incubate at 35 ° C for 4 days and pass several times until the number of cells required for seeding of the main culture is obtained.
[0087] 本培養の培地には、 EX-CELL™ 302培地を改変した培地 (JRH社製；以下、改 変 EX— CELL™302培地と記す）に、 MTX (シグマ社製） 500nmolZL、 L—グル タミン (和光純薬社製） 1. 75gZLを添加した培地を用いた。フィード培地には、ァミノ 酸（Lーァラニン 0. 14gZL、 L—アルギニン一塩酸 0. 47gZL、 Lーァスパラギン一 水和物 0. 16gZL、 Lーァスパラギン酸 0. 17gZL、 L—シスチン二塩酸 0. 51g/L 、： L—グルタミン酸 0. 42g/L,： L グルタミン 7. 3g/L,グリシン 0. 17g/L,： L ヒ スチジン一塩酸二水和物 0. 24gZL、 L—イソロイシン 0. 59gZL、 L一口イシン 0. 5 9gZL、 L—リジン一塩酸 0. 82gZL、 L メチォニン 0. 17gZL、 L—フエ-ルァラ ニン 0. 37gZL、 L プロリン 0. 22g/ L—セリン 0. 24g/L, L—スレオ-ン 0. 5 3gZL、L トリプトファン 0. 09g/L、 L—チロシンニナトリウム二水和物 0. 58g/L 、 L—パリン 0. 53g/L)、ビタミン（d—ピオチン 0. 073mgZL、 D—パントテン酸力 ノレシクム 0. 022g/L,塩ィ匕 =fジン 0. 022g/L,葉酸 0. 022g/L, myo—イノシ卜ー ル 0. 040gZL、ナイァシンアミド 0. 022gZL、ピリドキサール塩酸 0. 022g/L,リ ボフラビン 0. 0022gZL、チアミン塩酸 0. 022gZL、シァノコノ ラミン 0. 073mg/ L)、リコンビナントヒトインスリン 0. 31g/L (jRH社製）、エタノールァミン 0. 025g/ L (シグマ-アルドリッチ社製）、 2—メルカプトエタノール 0. 0098gZL (シグマ-アルド リッチ社製）、大豆加水分解物 HY— SOY8g/L (クウェストインターナショナル社製） 、亜セレン酸ナトリウム 16. 8 gZL (シグマ-アルドリッチ社製）、コレステロール脂質 濃縮溶液 2mL/L (250 X水溶液、インビトロジェン社製）、エチレンジァミン四酢酸 第二鉄ナトリウム塩 0. 05g/L (シグマ-アルドリッチ社製)を含む、改変 EX— CELLT M302培地を用いた。 [0087] The medium for the main culture includes a medium modified from EX-CELL ™ 302 medium (manufactured by JRH; hereinafter referred to as modified EX—CELL ™ 302 medium), MTX (manufactured by Sigma) 500 nmolZL, L— Glutamine (Wako Pure Chemical Industries, Ltd.) 1. A medium supplemented with 75 gZL was used. The feed medium contained amino acid (L-alanine 0.14 gZL, L-arginine monohydrochloride 0.47 gZL, L-asparagine monohydrate 0.16 gZL, L-aspartic acid 0.17 gZL, L-cystine dihydrochloride 0.51 g / L. A: L-glutamic acid 0.42 g / L, L glutamine 7.3 g / L, glycine 0.17 g / L, L histidine monohydrochloride dihydrate 0.2 gZL, L-isoleucine 0.59 g ZL, L bite Ishin 0.5 9gZL, L-Lysine Monohydrochloride 0.82gZL, L Methionine 0.17gZL, L-Ferulalanine 0.37gZL, L Proline 0.22g / L-Serine 0.24g / L, L-Threo- 0.5 3gZL, L Tryptophan 0.09g / L, L-tyrosine disodium dihydrate 0.58g / L , L-parin 0.53 g / L), vitamins (d-piotin 0.073 mg ZL, D-pantothenic acid power Norecicum 0.02 g / L, salt 匕 = f gin 0.022 g / L, folic acid 0.022 g / L , myo-inosinol 0.040 gZL, niacinamide 0.022 gZL, pyridoxal hydrochloride 0.022 g / L, riboflavin 0 0022 gZL, thiamine hydrochloride 0.022 gZL, cyanoconoramine 0.073 mg / L), recombinant human insulin 0. 31g / L (jRH), ethanolamine 0.025g / L (Sigma-Aldrich), 2-mercaptoethanol 0.009gZL (Sigma-Aldrich), soy hydrolyzate HY— SOY8g / L (Quwest International), sodium selenite 16.8 gZL (Sigma-Aldrich), cholesterol lipid concentrated solution 2 mL / L (250 X aqueous solution, manufactured by Invitrogen), ethylenediamin tetraacetic acid ferric sodium salt 0. 05g / L (Sigma-Aldori Including company Ltd.), it was used a modified EX- CELL T M 302 medium.
[0088] 拡大培養により充分な細胞数を獲得したところで、 MTXを 500nmolZLで、 Lーグ ルタミンを 1. 75g/L含む、改変 EX— CELL™302培地 30mLを満たした 250mL 三角フラスコ（コ一-ング社製）に 3 X 105細胞 ZmLとなるように細胞を播種した。同 時にバルプロ酸 0. ImmolZLを培地に添カ卩した。 [0088] When a sufficient number of cells was obtained by expansion culture, a 250 mL Erlenmeyer flask (co--) filled with 30 mL of modified EX—CELL ™ 302 medium containing 500 nmol ZL of MTX and 1.75 g / L of L-glutamine was obtained. Cells) were seeded at 3 × 10 5 cells ZmL. At the same time, 0.1 ml of valproic acid was added to the medium.
培地へバルプロを添加後、 35°C、 100rpm、 5%COを吹き込んで 9日間培養した  After adding VALPRO to the medium, it was cultured for 9 days by blowing 35 ° C, 100rpm, 5% CO.
2  2
。同時に上記物質を添加しな 、培地での培養 (以下コントロールと記す)を行った。  . At the same time, culture in a medium (hereinafter referred to as control) was performed without adding the above substances.
[0089] 培養液を培養開始から 3、 5、 7および 9日目にそれぞれ採取し、生細胞密度 (細胞 ZmL)および生産された抗体の濃度 (mgZL)を測定した。なお、生細胞密度は 0. 4%トリパンブルー溶液 (インビトロジェン社製)を用いた色素排除法で、生産された 抗体の濃度 (以下、物質濃度ともいう。 )は HPLC (島津製作所社製)によってそれぞ れ測定した。 [0089] The culture broth was collected on days 3, 5, 7 and 9 from the beginning of the culture, and the viable cell density (cell ZmL) and the concentration of the produced antibody (mgZL) were measured. The viable cell density is a dye exclusion method using 0.4% trypan blue solution (manufactured by Invitrogen), and the concentration of the produced antibody (hereinafter also referred to as substance concentration) is determined by HPLC (manufactured by Shimadzu Corporation). Each was measured.
[0090] また、生細胞密度と経過時間の積の総和で累積細胞密度を示した。本実施例にお いては生細胞密度を培養開始から、 3、 5、 7、 9日目に測定し、累積細胞密度は測定 した生細胞密度を使用して以下の式 1より算出した。  [0090] The cumulative cell density was shown as the sum of the product of the viable cell density and the elapsed time. In this example, the viable cell density was measured on the third, fifth, seventh, and ninth days from the start of the culture, and the cumulative cell density was calculated from the following formula 1 using the measured viable cell density.
(式 1)  (Formula 1)
累積細胞密度 (細胞 ZmLZ日） = (播種細胞密度 +測定細胞密度） X経過時間 ÷ 2 また、比生産速度は以下の式 2より算出した。 Cumulative cell density (cell ZmLZ day) = (seeding cell density + measured cell density) X elapsed time ÷ 2 The specific production rate was calculated from the following formula 2.
(式 2)  (Formula 2)
比生産速度 (pgZ細胞 Z日） =物質濃度 (mgZL) ÷累積細胞密度 (細胞 ZmLZ 曰）  Specific production rate (pgZ cell Z day) = substance concentration (mgZL) ÷ cumulative cell density (cell ZmLZ 曰)
生存率は培養開始力も培養 9日目まで 80%以上の良好な値を示し 14日目にはコ ントロール培養では 82%であったのに対し、バルプロ酸を添カ卩した培養は 94%であ つた。到達最大細胞密度は、コントロール培養では培養 7日目に 4. 5 X 106細胞 Z 日であったのに対し、バルプロ酸を添カ卩した培養では培養 7日目に 1. 8 X 106細胞 /日であった。 The viability showed a good value of 80% or more until the ninth day of culture, and the viability was 82% in the control culture on the 14th day, whereas it was 94% in the culture supplemented with valproic acid. Atsuta. The maximum cell density reached was 4.5 x 10 6 cells on day 7 in the control culture, whereas 1.8 x 10 6 on day 7 in the culture supplemented with valproic acid. Cells / day.
[0091] 式 2で算出される抗体の比生産速度は、バルプロ酸を添加するとコントロール比の 3 倍まで増大した。なお、増殖が抑制されることから力価の増大は 1. 4倍であった。 実施例 2  [0091] The specific production rate of the antibody calculated by Equation 2 increased to 3 times the control ratio when valproic acid was added. The increase in titer was 1.4 times because growth was suppressed. Example 2
[0092] 抗体の無血清フ ドバツチ培養  [0092] Serum-free food culture of antibodies
抗 CD20抗体を生産する Ms704— CD20株（FERM BP— 10092)を用いて以 下のフエドバッチ培養を行った。  Using the Ms704-CD20 strain (FERM BP-10092) that produces anti-CD20 antibody, the following fed-batch culture was performed.
本培養までの拡大培養用培地には、 EX-CELL™ 302培地 (JRH社製）に、 Met hotrexate (以下、 MTXと記す） 500nmol/L (シグマアルドリッチ社製）、 L—グルタ ミン（和光純薬社製） 1. 75gZLを添カ卩した培地を用いた。 125mL、 250mLまたは 1 OOOmL容量の三角フラスコ（コ一-ング社製）に約 10〜30%量の上記培地を入れ、 3 X 105細胞 ZmLとなるように Ms704— CD20株の細胞懸濁液を播種した。 35°C で 4日間培養し、本培養の播種に必要な細胞数が獲得されるまで複数回継代した。 The medium for expansion until the main culture is EX-CELL ™ 302 medium (manufactured by JRH), Met hotrexate (hereinafter referred to as MTX) 500 nmol / L (manufactured by Sigma Aldrich), L-glutamine (Jun Wako) 1. A medium supplemented with 75 g ZL was used. Place approximately 10-30% of the above medium in a 125 mL, 250 mL, or 1 OOOmL Erlenmeyer flask (manufactured by Corning), and a cell suspension of Ms704-CD20 strain to 3 X 10 5 cells ZmL Sowing. 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.
[0093] 本培養の基本培地には、 EX-CELL™ 302培地を改変した培地 (JRH社製；以 下、改変 EX— CELL™302培地と記す）に、 MTX (シグマ社製） 500nmolZL、 L —グルタミン (和光純薬社製） 1. 75g/Lを添加した培地を用いた。フィード培地には ゝアミノ酸（L—ァラニン 0. 14g/ L—アルギニン一塩酸 0. 47g/ L—ァスパラ ギン一水和物 0. 16gZL、 Lーァスパラギン酸 0. 17gZL、 L—シスチン二塩酸 0. 5 lg/ L—グルタミン酸 0. 42g/L、 L—グルタミン 7. 3g/L、グリシン 0. 17g/L 、 L—ヒスチジン一塩酸二水和物 0. 24gZL、 L—イソロイシン 0. 59gZL、L—ロイ シン 0. 59gZL、 L ジジン一塩酸 0. 82gZL、： L—メチォニン 0. 17gZL、 L—フエ -ルァラ-ン 0. 37gZL、 L プロリン 0. 22gZL、 L セリン 0. 24g/L, L—スレオ ニン 0. 53gZL、 L トリプトファン 0. 09g/L、 L—チロシンニナトリウム二水和物 0. 58gZL、 L—ノ リン 0. 53g/L)、ビタミン（d—ピオチン 0. 073mg/L、 D—パント テン酸カノレシクム 0. 022g/L,塩ィ匕 =3ジン 0. 022g/L,葉酸 0. 022g/L, myo— イノシトール 0. 040gZL、ナイァシンアミド 0. 022gZL、ピリドキサール塩酸 0. 022 g/L、リボフラビン 0. 0022g/L、チアミン塩酸 0. 022g/L、シァノ =fノラミン 0. 07 3mgZL)、リコンビナントヒトインスリン 0. 31g/L(jRH社製）、エタノールァミン 0. 0 25gZL (シグマ-アルドリッチ社製）、 2—メルカプトエタノール 0. 0098gZL (シグマ -アルドリッチ社製）、大豆加水分解物 HY— SOY8gZL (クウエストインターナショナ ル社製）、亜セレン酸ナトリウム 16. 8 μ g/L (シグマ-アルドリッチ社製）、コレステロ ール脂質濃縮溶液 2mLZL (250 X水溶液、インビトロジェン社製）、エチレンジアミ ン四酢酸第二鉄ナトリウム塩 0. 05gZL (シグマ-アルドリッチ社製)を含む、改変 EX — CELL™302培地を用いた。 [0093] The basic medium of the main culture includes a modified medium of EX-CELL ™ 302 medium (manufactured by JRH; hereinafter referred to as modified EX-CELL ™ 302 medium), MTX (manufactured by Sigma) 500 nmolZL, L —Glutamine (Wako Pure Chemical Industries, Ltd.) 1. A medium supplemented with 75 g / L was used. The feed medium contained ゝ amino acids (L-alanine 0.14 g / L-arginine monohydrochloride 0.47 g / L-asparagine monohydrate 0.16 gZL, L-aspartic acid 0.17 gZL, L-cystine dihydrochloride 0.5 lg / L-glutamic acid 0.42 g / L, L-glutamine 7.3 g / L, glycine 0.17 g / L, L-histidine monohydrochloride dihydrate 0.24 gZL, L-isoleucine 0.59 gZL, L-leu Shin 0.59gZL, L Dizine Monohydrochloride 0.82gZL, L-methionine 0.17gZL, L-Hue-Lulan 0.37gZL, L Proline 0.22gZL, L Serine 0.24g / L, L-Threonine 0.5gZL, L tryptophan 0.09g / L, L-tyrosine disodium dihydrate 0.58gZL, L-noline 0.53g / L), vitamin (d-piotine 0.073mg / L, D-pant Canorecicum Tenate 0.02 g / L, salt 匕 = 3 gin 0. 022 g / L, folic acid 0. 022 g / L, myo—inositol 0. 040 gZL, niacinamide 0.02 gZL, pyridoxal HCl 0.022 g / L, riboflavin 0.0022 g / L, thiamine hydrochloride 0.022 g / L, cyan = f noramine 0.07 3 mgZL, recombinant human insulin 0.31 g / L (manufactured by jRH), ethanolamine 0.0 25 gZL (Sigma-Aldrich) 2) Mercaptoethanol 0.009gZL (Sigma-Aldrich), soy hydrolyzate HY—SOY8gZL (Queus International) ), Sodium selenite 16.8 μg / L (Sigma-Aldrich), cholesterol lipid concentrated solution 2 mLZL (250 X aqueous solution, manufactured by Invitrogen), sodium ethylenediaminetetraacetic acid ferric acid sodium salt Modified EX — CELL ™ 302 medium containing 0.05 gZL (Sigma-Aldrich) was used.
[0094] 拡大培養により充分な細胞数を獲得したところで、 MTXを 500nmolZLで、 Lーグ ルタミンを 1. 75g/L含む、改変 EX—CELL™302培地 30mLを満たした 250 mL 三角フラスコ（コ一-ング社製）に 3 X 105細胞 ZmLとなるように細胞を播種した。同 時に表 1に記載の各種物質を、培地にそれぞれ添加した。 [0094] When a sufficient number of cells were obtained by expansion culture, a 250 mL Erlenmeyer flask (co-coil) filled with 30 mL of modified EX—CELL ™ 302 medium containing 500 nmol ZL of MTX and 1.75 g / L of L-glutamine was obtained. Cells were seeded at 3 × 10 5 cells ZmL. At the same time, various substances listed in Table 1 were added to the medium.
[0095] [表 1]  [0095] [Table 1]
Figure imgf000028_0001
Figure imgf000028_0001
培地へ各種物質を添加後、 35°C、 100rpm、 5%COを吹き込んで 14日間培養し  After adding various substances to the medium, incubate at 35 ° C, 100 rpm, 5% CO for 14 days.
2  2
た。培養開始から、 3、 5、 7、 9および 11日目に初発培地量の 8. 3%のフィード培地 を添加した。また、培養開始から 3日目以降においては、グルコース濃度が約 4gZL となるよう〖こ、 200g/Lグルコース溶液を適宜添カ卩した。同時に上記物質を添カ卩しな V、培地での培養 (以下、コントロールと記す)を行った。 It was. On the third, fifth, seventh, ninth and eleventh days from the beginning of the culture, 8.3% of the initial culture medium was added to the feed medium. Also, after the third day from the start of culture, the glucose concentration is about 4 gZL. Then, 200 g / L glucose solution was appropriately added. At the same time, the above-mentioned substances were cultured with V and medium (hereinafter referred to as control) without supplementation.
[0097] 培養液を培養開始から 3、 5、 7、 9、 11および 14日目にそれぞれ採取し、生細胞密 度 (細胞 ZmL)および生産された抗体の濃度 (mgZL)を測定した。なお、生細胞密 度は 0. 4%トリパンブルー溶液 (インビトロジェン社製)を用いた色素排除法で、生産 された抗体の濃度 (以下、物質濃度ともいう。 )は HPLC (島津製作所社製)によって それぞれ測定した。累積細胞密度と比生産速度は実施例 1と同様にして算出した。  [0097] Culture fluids were collected on days 3, 5, 7, 9, 11 and 14 from the start of culture, and the viable cell density (cell ZmL) and the concentration of the produced antibody (mgZL) were measured. The density of live cells is determined by dye exclusion using a 0.4% trypan blue solution (Invitrogen). The concentration of the antibody produced (hereinafter also referred to as the substance concentration) is HPLC (manufactured by Shimadzu Corporation). Respectively. The cumulative cell density and specific production rate were calculated in the same manner as in Example 1.
[0098] 表 1に記載される物質の添カ卩によって、抗体濃度はコントロール対比 104〜145% に増大した。抗体の比生産速度は図 1から明らかなように表に示した全ての物質にお いて、比生産速度はコントロールと比較して 127〜239%の生産性を示した。  [0098] By adding the substances listed in Table 1, the antibody concentration was increased to 104 to 145% of the control. As is clear from FIG. 1, the specific production rate of the antibody showed a productivity of 127 to 239% in all substances shown in the table as compared with the control.
実施例 3  Example 3
[0099] Ms705ZAT— III株を用いる ΑΤΠΙ (アンチトロンビン III)の無血清フエドバッチ培養  [0099] Serum-free fed-batch culture of ΑΤΠΙ (antithrombin III) using Ms705ZAT—III strain
ATIII (アンチトロンビン III)を生産する能力を有する、 Ms705-pKAN— ΑΤΠΙ株（ FERM-BP8472)を用いて以下のフエドバッチ培養を行った。  The following fed-batch culture was performed using Ms705-pKAN-ΑΤΠΙ strain (FERM-BP8472) having the ability to produce ATIII (antithrombin III).
本培養までの拡大培養用培地には、 EX— CELL™ 302培地 (JRH社製)を用い た。 EX—CELL™302培地には Methotrexate (以下、 MTXと記す） 500nmolZL (シグマアルドリッチ社製）、 L—グルタミン (和光純薬社製） 1. 75gZLを添加した。 1 25mL、 250mLまたは lOOOmL容量の三角フラスコ（コ一-ング社製）に約 10〜30 %量の上記培地を入れ、 3 X 105細胞 ZmLとなるように Ms705_pKAN— ΑΤΠΙ株（ FERM— BP8472)の細胞懸濁液を播種した。 35°Cで 4日間培養し、本培養の播種 に必要な細胞数が獲得されるまで複数回継代した。 EX-CELL ™ 302 medium (manufactured by JRH) was used as the medium for expansion until the main culture. To EX-CELL ™ 302 medium, Methotrexate (hereinafter referred to as MTX) 500 nmolZL (Sigma Aldrich) and L-glutamine (Wako Pure Chemical Industries) 1.75 gZL were added. 1 25 mL, 250 mL or lOOOmL Erlenmeyer flask - put the medium to about 10-30% amount (co one ring Co.), such that the 3 X 10 5 cells ZmL Ms705_pKAN- ΑΤΠΙ strain (FERM- BP8472) Cell suspension. 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.
[0100] 本培養の基本培地には、 EX-CELL™ 302培地を改変した培地 (JRH社製；以 下、改変 EX— CELL™302培地と記す）を用いた。本培養では、改変 EX— CELLT M302培地に、 MTX (シグマ社製） 500nmolZL、 L—グルタミン（和光純薬社製） 1. 75g/Lを添カ卩した。フィード培地には、アミノ酸 (L—ァラニン 0. 14g/L、 L—アル ギニン一塩酸 0. 47gZL、 Lーァスパラギン一水和物 0. 16gZL、 Lーァスパラギン 酸 0. 17gZL、 L—シスチン二塩酸 0. 51gZL、 L—グルタミン酸 0. 42g/L, L—グ ルタミン 7. 3gZL、グリシン 0. 17gZL、 L—ヒスチジン一塩酸二水和物 0. 24g/L 、L—イソロイシン 0. 59g/L、 L—ロイシン 0. 59gZL、 L—リジン一塩酸 0. 82g/L 、： L—メチォニン 0. 17gZL、： L—フエ-ルァラニン 0. 37gZL、： L—プロリン 0. 22g 7しュ—セリン0. 24gZL、 L—スレオ-ン 0. 53gZL、 L—トリプトファン 0. 09g/L 、 L—チロシンニナトリウム二水和物 0. 58gZL、 Lーノ リン 0. 53gZL)、ビタミン（d —ピオチン 0. 073mgZL、 D—パントテン酸カルシウム 0. 022g/L,塩化コリン 0. 022gZL、葉酸 0. 022gZL、 myo—イノシトール 0. 040g/L,ナイァシンアミド 0. 022g/L,ピリドキサール塩酸 0. 022g/L,リボフラビン 0. 0022g/L,チアミン塩 酸 0. 022gZL、シァノコバラミン 0. 073mg/L)、リコンビナントヒトインスリン 0. 31g /L (JRH社製）、エタノールァミン 0. 025g/L (シグマ-アルドリッチ社製）、 2—メル カプトエタノール 0. 0098g/L (シグマ-アルドリッチ社製）、大豆カ卩水分解物 HY—S OY8gZL (クウェストインターナショナル社製）、亜セレン酸ナトリウム 16. 8 gZL ( シグマ-アルドリッチ社製）、コレステロール脂質濃縮溶液 2mLZL (250 X水溶液、 インビトロジェン社製）、エチレンジァミン四酢酸第二鉄ナトリウム塩 0. 05g/L (シグ マ-アルドリッチ社製)をそれぞれ含む、培地を用いた。 [0100] A medium obtained by modifying EX-CELL ™ 302 medium (manufactured by JRH; hereinafter referred to as modified EX-CELL ™ 302 medium) was used as the basic medium for the main culture. In the main culture, MTX (Sigma) 500 nmolZL and L-glutamine (Wako Pure Chemical Industries) 1.75 g / L were added to the modified EX-CELL T M 302 medium. The feed medium contained amino acids (L-alanine 0.14 g / L, L-arginine monohydrochloride 0.47 gZL, L-parasparagin monohydrate 0.16 gZL, L-parasine acid 0.17 gZL, L-cystine dihydrochloride 0. 51 gZL, L-glutamic acid 0.42 g / L, L-glutamine 7.3 gZL, glycine 0.17 gZL, L-histidine monohydrochloride dihydrate 0.24 g / L , L-isoleucine 0.59g / L, L-leucine 0.59gZL, L-lysine monohydrochloride 0.82g / L, L-methionine 0.17gZL, L-felualanine 0.37gZL, L-proline 0.22 g 7 μserine 0.24 gZL, L-threonine 0.53 gZL, L-tryptophan 0.09 g / L, L-tyrosine disodium dihydrate 0.58 gZL, L-noline 0.53 gZL ), Vitamin (d—Piotin 0.073mgZL, D—Calcium pantothenate 0.02g / L, Choline chloride 0.022gZL, Folic acid 0.022gZL, myo—Inositol 0.040g / L, Niacinamide 0.022g / L, Pyridoxal Hydrochloric acid 0.022 g / L, Riboflavin 0.0022 g / L, Thiamine hydrochloride 0.022 g ZL, cyanobalamine 0.073 mg / L), Recombinant human insulin 0.31 g / L (manufactured by JRH), Ethanolamine 025g / L (Sigma-Aldrich), 2-mercaptoethanol 0.009g / L (Sigma-Aldrich), Soybean water decomposition HY-S OY8gZL (Quwest International), sodium selenite 16.8 gZL (Sigma-Aldrich), cholesterol lipid concentrated solution 2mLZL (250 X aqueous solution, manufactured by Invitrogen), sodium ethylenediammine tetraacetate A medium containing 0.05 g / L of salt (manufactured by Sigma-Aldrich) was used.
[0101] 拡大培養により充分な細胞数を獲得したところで、 （1R)— (+)—力ンファ (シグマァ ルドリッチ社製）をそれぞれ 0. 12mmolZL、0. 23mmol/L, 0. 39mmolZLまた は 0. 55 mmolZLで、 MTXを 500nmol/Lで、 L—グノレタミンを 1. 75gZLでそれ ぞれ含む、改変 EX— CELL™ 302培地 30mLを満たした 250 mL三角フラスコ（コ 一-ング社製）に 3 X 105細胞/ mLとなるように細胞を播種した。その後 35°C、 100r pm、 5%COを吹き込んで 14日間培養した。培養開始力も、 3、 5、 7、 9および 11日 [0101] When a sufficient number of cells were obtained by expansion culture, (1R) — (+) — Strength (manufactured by Sigma Aldrich) was added to 0.12 mmolZL, 0.23 mmol / L, 0.39 mmolZL, or 0.30 mmol, respectively. In a 250 mL Erlenmeyer flask (Corning) filled with 30 mL of modified EX—CELL ™ 302 medium containing 55 mmolZL, MTX at 500 nmol / L, and L-gnoretamine at 1.75 gZL, 3 X Cells were seeded to 10 5 cells / mL. Thereafter, the cells were cultured for 14 days while blowing 35 ° C, 100 rpm, and 5% CO. Incubation force is also 3, 5, 7, 9 and 11 days
2  2
目に初発培地量の 8. 3%のフィード培地を添加した。また、培養開始から 3日目以降 においては、グルコース濃度が約 4g/Lとなるように、 200g/Lグルコース溶液を適宜 添加した。同時にカンファを含まな!/、培地での培養 (以下コントロールと記す)を行つ た。  To the eye, 8.3% of the initial medium amount of feed medium was added. In addition, from the third day after the start of culture, a 200 g / L glucose solution was appropriately added so that the glucose concentration was about 4 g / L. At the same time, no camphor was included! /, And culture in medium (hereinafter referred to as control) was performed.
[0102] カンファを培地に添加する際には、 DMSO (シグマアルドリッチ社製） 1 mLにカン ファ 334. 9 mgの割合で溶解したカンファ濃縮液を用いた。  [0102] When adding camphor to the medium, a camphor concentrate dissolved in a ratio of 334.9 mg of camphor in 1 mL of DMSO (manufactured by Sigma-Aldrich) was used.
培養液を培養開始から 3、 5、 7、 9、 11および 14日目にそれぞれ採取し、生細胞密 度 (細胞 ZmL)および生産された ΑΤΠΙの濃度 (mgZL)を測定した。なお、生細胞 密度は 0. 4%トリパンブルー溶液 (インビトロジェン社製)を用いた色素排除法で、生 産された ΑΤΠΙの濃度 (以下、物質濃度ともいう。 )は HPLC (島津製作所社製）によつ てそれぞれ測定した。 The culture broth was collected on days 3, 5, 7, 9, 11, and 14 from the beginning of the culture, and the viable cell density (cell ZmL) and the concentration of sputum produced (mgZL) were measured. Live cells Density is a dye exclusion method using 0.4% trypan blue solution (manufactured by Invitrogen). The concentration of soot produced (hereinafter also referred to as substance concentration) is determined by HPLC (manufactured by Shimadzu Corporation). Each was measured.
[0103] また、生細胞密度と経過時間の積の総和で累積細胞密度を示した。本実施例にお いては生細胞密度を培養開始から、 3、 5、 7、 9、 11および 14日目に測定し、累積細 胞密度は測定した生細胞密度を使用して以下の式 3により算出した。  [0103] The cumulative cell density was expressed as the sum of the product of the viable cell density and the elapsed time. In this example, the viable cell density was measured on the third, fifth, seventh, ninth, eleventh, and fourteenth days from the start of the culture, and the cumulative cell density was calculated using the measured viable cell density using the following formula 3 Calculated by
(式 3)  (Formula 3)
累積細胞密度 (細胞 ZmLZ日） = (培養 0日目細胞密度 +培養 3日目細胞密度) / 2 （3— 0)日+ (培養3日目細胞密度+培養5日目細胞密度)72 （5— 3)日+ ' · · + (培養 11日目細胞密度 +培養 14日目細胞密度) Z2 X ( 14—11)日 比生産速度は、培養の終了時点に培養上清に蓄積された物質の濃度 (mgZL)を 累積細胞密度 (細胞 ZmLZ日）で除することにより算出した。  Cumulative cell density (cell ZmLZ day) = (culture day 0 cell density + culture day 3 cell density) / 2 (3—0) day + (culture day 3 cell density + culture day 5 cell density) 72 ( 5-3) day + '· · + (cell density on day 11 of culture + cell density on day of culture 14) Z2 X (14–11) day The specific production rate was accumulated in the culture supernatant at the end of the culture The substance concentration (mgZL) was calculated by dividing by the cumulative cell density (cell ZmLZ day).
[0104] 生存率は培養開始力も培養 10日目まで 90%以上の良好な値を示し 14日目には コントロール培養では 79%であったのに対し、カンファを添カロした培養は 91 %であつ た。到達最大細胞密度は、コントロール培養では培養 11日目に 7. 4 X 106細胞 Z日 であったのに対し、カンファを 0. 55mmol/Lの濃度で添カロした培養では培養 14日 目に 7. 4 X 106細胞 Z日であった。 [0104] The viability showed a good value of 90% or more until the 10th day of culture, and the viability was 79% in the control culture on the 14th day, whereas 91% in the culture supplemented with camphor. It was hot. The maximum cell density reached was 7.4 x 10 6 cells Z on day 11 in the control culture, whereas on day 14 in culture supplemented with camphor at a concentration of 0.55 mmol / L. 7. 4 × 10 6 cells Z days.
[0105] 式 3で算出される ΑΤΠΙの比生産速度をカンファ添加濃度毎にそれぞれ図 2に示し た。 ΑΤΠΙの比生産速度は、 0. 55mmolZLの濃度でカンファを添カ卩した場合が最 大になった。  [0105] The specific production rate of koji calculated by Equation 3 is shown in Fig. 2 for each camphor addition concentration. The specific production rate of soot was maximized when camphor was added at a concentration of 0.55 mmol ZL.
以上のことから、 Ms705/AT— III株の三角フラスコを用いた培養において、カン ファを添加することにより AT— IIIの生産性が向上することが確認された。  From the above, it was confirmed that the productivity of AT-III was improved by adding a camphor in the culture using the Erlenmeyer flask of Ms705 / AT-III strain.
[0106] また、培養開始から 11日目と 14日目での累積細胞密度 (細胞/ mL X日）に対する 生産された ΑΤΠΙの濃度 (mg/L)を図 3に示した。累積細胞密度は、コントロール培 養では 14日間で 5. 8 X 106細胞 ZmLZ日であり、生産された ΑΤΠΙの濃度は 985 mgZLであったことから比生産速度は 170pgZ細胞 Z日であった。一方、カンファを 0. 55mmol/Lの濃度で添加した培養では累積生細胞密度は、 14日間で 4. 3 X 1 06細胞 ZmLZ日であり、生産された ΑΤΠΙの濃度は 1078 mgZLであったことから、 比生産速度は 251pgZ細胞 Z日であった。 [0106] Fig. 3 shows the concentration (mg / L) of sputum produced relative to the cumulative cell density (cells / mL x day) on day 11 and day 14 after the start of culture. The cumulative cell density was 5.8 × 10 6 cells ZmLZ day in 14 days in the control culture, and the concentration of sputum produced was 985 mgZL, so the specific production rate was 170 pgZ cell Z days. On the other hand, in the culture added with camphor at a concentration of 0.55 mmol / L, the cumulative viable cell density was 4.3 X 10 6 cells ZmLZ day in 14 days, and the concentration of sputum produced was 1078 mgZL From that The specific production rate was 251 pgZ cells Z days.
[0107] このように、細胞あたりの物質の比生産速度は、カンファの濃度と相関して増大する 力 累積細胞密度はカンファの濃度と逆相関して減少する。従って、添加するカンフ ァの濃度は、生産に用いる細胞の増殖と比生産速度とを考慮して設定する必要があ ることが明ら力となった。 [0107] Thus, the specific production rate of substances per cell increases in correlation with the concentration of camphor. The cumulative cell density decreases inversely with the concentration of camphor. Therefore, it has become clear that the concentration of the added camphor must be set in consideration of the growth of cells used for production and the specific production rate.
実施例 4  Example 4
[0108] 抗体生産細胞株を用いる無血清フエドバッチ培養  [0108] Serum-free fed-batch culture using antibody-producing cell lines
Ms705- pKAN— ΑΤΠΙ株の代わりに、抗 CD20抗体を生産する Ms704— CD20 株（FERM BP— 10092)および抗 CCR4ヒト化抗体を生産する 709 LCA—500G 株を用いる以外は実施例 3に記載の方法と同様にしてフエドバッチ培養を実施した。 ただし、カンファは 0. 55mmol/Lの濃度で添カ卩した。尚、 709 LCA— 500G株は 、 WO03Z046174の実施例 3に記載の 503LCA— 500D株と同様の方法により取 得した。  Instead of the Ms705-pKAN—pox strain, the Ms704—CD20 strain producing the anti-CD20 antibody (FERM BP-10092) and the 709 LCA-500G strain producing the anti-CCR4 humanized antibody were used as described in Example 3. The fed-batch culture was performed in the same manner as the method. However, camphor was added at a concentration of 0.55 mmol / L. The 709 LCA-500G strain was obtained in the same manner as the 503LCA-500D strain described in Example 3 of WO03Z046174.
[0109] その結果、 Ms704— CD20株の三角フラスコを用いた培養において、コントロール 培養では、培養開始から 14日目においての累積細胞密度は、 5. 8 X 106細胞/ m LZ日であり、抗体濃度は 985 mgZLであったことから比生産速度は 170pgZ細胞 /日であったのに対し、カンファを添カロした培養では、培養開始から 14日目におい ての累積生細胞密度は 4. 3 X 106細胞 ZmLZ日であり、生産された抗体の濃度は 1078 mgZLであったこと力 比生産速度は 251pgZ細胞 Z日であった。培養開始 力 14日目での、カンファの添加または非添カ卩時の培養における、生産された抗 C D20抗体の比生産速度を図 4に示した。 As a result, in the culture using the Erlenmeyer flask of Ms704-CD20 strain, in the control culture, the cumulative cell density on the 14th day from the start of the culture was 5.8 × 10 6 cells / mLZ, Since the antibody concentration was 985 mgZL, the specific production rate was 170 pgZ cells / day, whereas in the culture supplemented with camphor, the cumulative viable cell density on the 14th day from the start of culture was 4.3. X 10 6 cells were ZmLZ days, and the concentration of antibody produced was 1078 mgZL, and the specific production rate was 251 pgZ cells Z days. Fig. 4 shows the specific production rate of the produced anti-CD20 antibody in the culture at the start of the culture on day 14 with or without camphor.
[0110] また、 709 LCA— 500G株の三角フラスコを用いた培養においても、コントロール 培養では、培養開始から 14日目において累積細胞密度は、 4. 7 X 106細胞/ mL Z日であり、抗体濃度は 590 mgZLであったことから比生産速度は 126pgZ細胞 /日であったのに対し、カンファを添カロした培養では、培養開始から 14日目におい て累積生細胞密度は 2. O X 106細胞 ZmLZ日であり、抗体濃度は 425 mgZLで あったことから比生産速度は 213pgZ細胞 Z日であった。培養開始から 14日目での 、カンファの添加または非添カ卩時の培養における、生産された抗 CCR4ヒト化抗体の 比生産速度を図 5に示した。 [0110] Also, in the culture using the 709 LCA-500G Erlenmeyer flask, in the control culture, the cumulative cell density was 4.7 X 10 6 cells / mL Z day on the 14th day from the start of the culture. Since the antibody concentration was 590 mgZL, the specific production rate was 126 pgZ cells / day, whereas in the culture supplemented with camphor, the cumulative viable cell density was 2.OX 10 on the 14th day from the start of culture. Since the 6- cell ZmLZ day and the antibody concentration was 425 mgZL, the specific production rate was 213 pgZ-cell Z-day. On the 14th day after the start of culture, the anti-CCR4 humanized antibody produced in the culture with or without camphor was added. The specific production rate is shown in Fig. 5.
[0111] 以上のことから、実施例 3で確認された ΑΤΠΙ産生細胞以外に、遺伝子組換え抗体 を生産する形質転換細胞においても、カンファの添カ卩による生産性向上効果が確認 された。 [0111] From the above, in addition to the cocoon-producing cells confirmed in Example 3, the productivity-improving effect of camphor supplementation was also confirmed in transformed cells that produced recombinant antibodies.
実施例 5  Example 5
[0112] カンファの添カ卩時期による生産性向上効果の検討  [0112] Examination of productivity improvement effect by camphor dressing time
抗 CD20抗体を生産する Ms704— CD20株（FERM BP— 10092)を用いて、実 施例 4と同様にしてフヱドバッチ培養を行った。  Food batch culture was performed in the same manner as in Example 4 using Ms704-CD20 strain (FERM BP-10092) that produces anti-CD20 antibody.
†dt 、カンファ ίま 0. 55mmol/:Lの濃度で、 0、 3、 5、 7、 9および 11曰目にフィー ド培地と同時にそれぞれ添加した。  † Dt and camphor were added at a concentration of 0.555 mmol /: L at 0, 3, 5, 7, 9 and 11 at the same time as the feed medium.
[0113] 図 6に、カンファを添加した時期毎の、培養中の生細胞数を示した。細胞の生存率 は培養開始力 培養 10日目まで 90%以上の良好な値を示し 14日目にはコントロー ル培養では 79%であったのに対し、培養開始力もカンファを添加した培養は 91%で あった。到達最大密度はコントロール培養では培養開始から 11日目において 7. 4 X 106細胞 Z日であったのに対し、カンファを添加した培養では、培養開始から 14日目 において 7. 4 X 106細胞/日であり、カンファを培養の初期に添加することで、到達 最大密度に達するまでの培養日数が延長されたことから、カンファの添カ卩により細胞 の増殖が抑制されていることが明ら力となった。 [0113] Fig. 6 shows the number of viable cells in culture for each period when camphor was added. The viability of the cells showed a good value of 90% or more until the 10th day of culture, which was 79% in the control culture on the 14th day, whereas the culture start power was 91% in the culture with the addition of camphor. %Met. The maximum density reached in the control culture was 7.4 x 10 6 cells on day 11 from the start of culture, whereas in the culture with camphor added, it was 7.4 x 10 6 on day 14 from the start of culture. Since the number of days to reach the maximum density was extended by adding camphor at the initial stage of culture, the cell growth was suppressed by the addition of camphor. It became power.
[0114] 図 7に、カンファを添加した時期毎の、物質の比生産速度を示した。図 7に示される ように、抗体の比生産速度は、培養開始と同時にカンファを添加した場合が最も高く 、培養開始から 5日目までにカンファを添加した場合には、カンファ添カ卩による抗体 の生産性向上効果が認められた。一方、培養開始から 7日目以降にカンファを添カロ した場合には、カンファ添カ卩による、顕著な抗体の生産性向上効果は認められなか つた o  [0114] Fig. 7 shows the specific production rate of the substance at each time when camphor was added. As shown in Fig. 7, the specific antibody production rate was highest when camphor was added at the same time as the start of culture, and when camphor was added within 5 days from the start of culture, The productivity improvement effect was recognized. On the other hand, when camphor was added after 7 days from the start of culture, no significant improvement in antibody productivity was observed.
[0115] 以上のことから、カンファを培地に添カ卩して抗体などの物質を培養して生産する場 合には、培養の初期、即ち細胞が活発に増殖している時期にカンファを添加するの 1S 比生産速度の向上という点では、好ましいことが示された力 同時に培養の初期 にカンファを添加すると細胞の増殖が抑制されてしまうことが明ら力となった。従って、 カンファを培地に添加時期は、生産に用いる細胞の増殖と比生産速度とを考慮して 設定する必要がある。 [0115] From the above, when camphor is added to a culture medium and a substance such as an antibody is cultured for production, the camphor is added at the initial stage of the culture, that is, when the cells are actively proliferating. However, in terms of improving the 1S specific production rate, it has been shown that it is preferable. At the same time, the addition of camphor at the initial stage of the culture suppressed cell growth. Therefore, The timing for adding camphor to the medium needs to be set in consideration of the growth of cells used for production and the specific production rate.
実施例 6  Example 6
[0116] 709 LCA— 500G株を用いる抗 CCR4ヒト型キメラ抗体の無血清フエドバッチ培養 抗 CCR4ヒト型キメラ抗体を生産する能力を有する、 709 LCA— 500G株を用いて 以下のフエドバッチ培養を行った。尚、 709 LCA— 500G株は、 WO03Z046174 の実施例 3に記載の 503 LCA—500D株（FERM BP— 8239)と同様の方法に より取得した。  Serum-free fed-batch culture of anti-CCR4 human chimeric antibody using 709 LCA-500G strain The following fed-batch culture was performed using 709 LCA-500G strain having the ability to produce anti-CCR4 human chimeric antibody. The 709 LCA-500G strain was obtained by the same method as the 503 LCA-500D strain (FERM BP-8239) described in Example 3 of WO03Z046174.
[0117] 本培養までの拡大培養用培地には、 EX—CELL™ 302培地 (JRH社製）を用い た。 EX—CELL™302培地には Methotrexate (以下、 MTXと記す） 500nmolZL (シグマアルドリッチ社製）、 L—グルタミン (和光純薬社製） 1. 75gZLを添加した。 1 25mL、 250mLまたは lOOOmL容量の三角フラスコ（コ一-ング社製）に約 10〜30 %量の上記培地を入れ、 3 X 105細胞 ZmLとなるように 709 LCA— 500G株の細 胞懸濁液を播種した。 35°Cで 4日間培養し、本培養の播種に必要な細胞数が獲得さ れるまで複数回継代した。 [0117] EX-CELL ™ 302 medium (manufactured by JRH) was used as the medium for expansion until the main culture. To EX-CELL ™ 302 medium, Methotrexate (hereinafter referred to as MTX) 500 nmolZL (Sigma Aldrich) and L-glutamine (Wako Pure Chemical Industries) 1.75 gZL were added. 1 Place approximately 10-30% of the above medium in a 25 mL, 250 mL or lOOOmL Erlenmeyer flask (manufactured by Corning Co., Ltd.), and 709 LCA-500G cell suspension to give 3 X 10 5 cells ZmL The suspension was seeded. 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.
[0118] 本培養の基本培地には、 EX-CELL™ 302培地を改変した培地 (JRH社製；以 下、改変 EX— CELL™302培地と記す）を用いた。本培養では、改変 EX— CELLT M302培地に、 MTX (シグマ社製） 500nmolZL、 L—グルタミン（和光純薬社製） 1. 75g/Lを添カ卩した。フィード培地には、アミノ酸 (L—ァラニン 0. 14g/L、 L—アル ギニン一塩酸 0. 47gZL、 Lーァスパラギン一水和物 0. 16gZL、 Lーァスパラギン 酸 0. 17gZL、 L—シスチン二塩酸 0. 51gZL、 L—グルタミン酸 0. 42g/L, L—グ ルタミン 7. 3gZL、グリシン 0. 17gZL、 L—ヒスチジン一塩酸二水和物 0. 24g/L 、L—イソロイシン 0. 59g/L、 L—ロイシン 0. 59gZL、 L—リジン一塩酸 0. 82g/L 、： L—メチォニン 0. 17gZL、： L—フエ-ルァラニン 0. 37gZL、： L—プロリン 0. 22g 7しュ—セリン0. 24gZL、 L—スレオ-ン 0. 53gZL、 L—トリプトファン 0. 09g/L 、 L—チロシンニナトリウム二水和物 0. 58gZL、 Lーノ リン 0. 53gZL)、ビタミン（d —ピオチン 0. 073mgZL、 D—パントテン酸カルシウム 0. 022g/L,塩化コリン 0. 022g/L,葉酸 0. 022gZL、 mvo—イノシトール 0. 040g/L,ナイァシンアミド 0. 022g/L,ピリドキサール塩酸 0. 022g/L,リボフラビン 0. 0022g/L,チアミン塩 酸 0. 022gZL、シァノコバラミン 0. 073mg/L)、リコンビナントヒトインスリン 0. 31g /L (JRH社製）、エタノールァミン 0. 025g/L (シグマ-アルドリッチ社製）、 2—メル カプトエタノール 0. 0098g/L (シグマ-アルドリッチ社製）、大豆カ卩水分解物 HY—S OY8gZL (クウェストインターナショナル社製）、亜セレン酸ナトリウム 16. 8 gZL ( シグマ-アルドリッチ社製）、コレステロール脂質濃縮溶液 2mLZL (250 X水溶液、 インビトロジェン社製）、エチレンジァミン四酢酸第二鉄ナトリウム塩 0. 05g/L (シグ マ-アルドリッチ社製)をそれぞれ含む、培地を用いた。 [0118] A medium obtained by modifying EX-CELL ™ 302 medium (manufactured by JRH; hereinafter referred to as modified EX-CELL ™ 302 medium) was used as the basic medium for the main culture. In the main culture, MTX (Sigma) 500 nmolZL and L-glutamine (Wako Pure Chemical Industries) 1.75 g / L were added to the modified EX-CELL T M 302 medium. The feed medium contained amino acids (L-alanine 0.14 g / L, L-arginine monohydrochloride 0.47 gZL, L-parasparagin monohydrate 0.16 gZL, L-parasine acid 0.17 gZL, L-cystine dihydrochloride 0. 51 gZL, L-glutamic acid 0.42 g / L, L-glutamine 7.3 gZL, glycine 0.17 gZL, L-histidine monohydrochloride dihydrate 0.24 g / L, L-isoleucine 0.59 g / L, L— Leucine 0.59 gZL, L-lysine monohydrochloride 0.82 g / L, L-methionine 0.17 gZL, L-fe-lualanine 0.37 gZL, L-proline 0.22 g 7 sucrose 0.24 gZL, L-threonine 0.53gZL, L-tryptophan 0.09g / L, L-tyrosine disodium dihydrate 0.58gZL, L-noline 0.53gZL), vitamin (d-piotin 0.073mgZL, D —Calcium pantothenate 0.02 g / L, choline chloride 0.022 g / L, folic acid 0.02 gZL, mvo—inositol 0.040 g / L, niacinamide 0. 022 g / L, pyridoxal hydrochloride 0.02 0 g / L, riboflavin 0.0022 g / L, thiamine hydrochloride 0.22 g ZL, cyanobalamine 0.073 mg / L), recombinant human insulin 0.31 g / L (manufactured by JRH), Ethanolamine 0.025g / L (Sigma-Aldrich), 2-Mercaptoethanol 0.009g / L (Sigma-Aldrich), Soybean cocoon hydrolyzate HY—S OY8gZL (Quwest International) , Sodium selenite 16.8 gZL (Sigma-Aldrich), Cholesterol lipid concentrated solution 2mLZL (250 X aqueous solution, Invitrogen), Ethylenediamin tetraacetic acid ferric sodium salt 0.05 g / L (Sigma-Aldrich) Medium) containing each of them was used.
[0119] 拡大培養により充分な細胞数を獲得したところで、それぞれ MTXを 500nmolZL で、 L—グルタミンを 1. 75gZLで添カ卩した、上記の改変 EX— CELL™302培地 30 mLを満たした 250 mL三角フラスコ（コ一-ング社製）に 3 X 105細胞 ZmLとなるよう に細胞を播種し、 35°C、 100rpm、 5%COを吹き込んで 14日間培養した。コウジ酸 [0119] When sufficient cell counts were obtained by expansion culture, 250 mL filled with 30 mL of the above modified EX-CELL ™ 302 medium supplemented with 500 nmolZL of MTX and 1.75 gZL of L-glutamine, respectively. Cells were seeded in an Erlenmeyer flask (manufactured by Corning Co., Ltd.) at 3 × 10 5 cells ZmL, and cultured at 35 ° C., 100 rpm, 5% CO for 14 days. Kojic acid
2  2
(シグマアルドリッチ社製）はそれぞれ 0. 5mmolZLまたは 1. 0 mmolZL2なるよう に培養開始から、 0、 3または 5日目に添加した。  (Manufactured by Sigma-Aldrich) was added at 0, 3 or 5 days from the start of the culture so as to be 0.5 mmolZL or 1.0 mmolZL2, respectively.
[0120] 培養開始から、 3、 5、 7、 9および 11日目に初発培地量の 8. 3%のフィード培地を 添加した。また、培養開始から 3日目以降においては、グルコース濃度が約 4gZLと なるように、 200g/Lグルコース溶液を適宜添加した。上記の培養と同時に、コウジ酸 を含まない培地での培養 (以下コントロールと記す)を行った。  [0120] On the third, fifth, seventh, ninth and eleventh days from the start of the culture, 8.3% of the initial culture medium was added. Further, after the third day from the start of the culture, a 200 g / L glucose solution was appropriately added so that the glucose concentration was about 4 gZL. Simultaneously with the above culture, culture in a medium not containing kojic acid (hereinafter referred to as control) was performed.
培養液を培養開始から 3、 5、 7、 9、 11、 13および 15日目にそれぞれ採取し、生細 胞密度 (細胞 ZmL)および生産された抗体の濃度 (mgZL)を測定した。なお、生細 胞密度は 0. 4%トリパンブルー溶液 (インビトロジェン社製)を用いた色素排除法で、 生産された抗体の濃度 (以下、物質濃度ともいう。 )は HPLC (島津製作所社製)によ つてそれぞれ測定した。  The culture medium was collected on days 3, 5, 7, 9, 11, 13, and 15 from the beginning of the culture, and the density of live cells (cell ZmL) and the concentration of produced antibody (mgZL) were measured. The density of live cells is a dye exclusion method using 0.4% trypan blue solution (manufactured by Invitrogen). The concentration of the antibody produced (hereinafter also referred to as substance concentration) is HPLC (manufactured by Shimadzu Corporation). Respectively.
[0121] また、生細胞密度と経過時間の積の総和で累積細胞密度を示した。本実施例にお いては生細胞密度を培養開始から、 3、 5、 7、 9、 11、 13および 15日目に測定し、累 積細胞密度は測定した生細胞密度を使用して以下の式 4により算出した。  [0121] The cumulative cell density was expressed as the sum of the product of the viable cell density and the elapsed time. In this example, the viable cell density was measured on the third, fifth, seventh, ninth, eleventh, thirteenth, and fifteenth days from the start of the culture, and the cumulative cell density was measured using the measured live cell density as follows. Calculated according to Equation 4.
(式 4)  (Formula 4)
累積細胞密度 (細胞 ZmLZ日） = (培養 0日目細胞密度 +培養 3日目細胞密度) / 2 （3— 0)日+ (培養3日目細胞密度+培養5日目細胞密度)72 （5— 3)日+ ' · · + (培養 13日目細胞密度 +培養 15日目細胞密度) Z2X (15— 13)日 比生産速度は、培養の終了時点に培養上清に蓄積された物質の濃度 (mgZL)を 累積細胞密度 (細胞 ZmLZ日）で除することにより算出した。 Cumulative cell density (cell ZmLZ day) = (culture day 0 cell density + culture day 3 cell density) / 2 (3-0) day + (culture day 3 cell density + culture day 5 cell density) 72 (5-3) day + '· · + (culture day 13 cell density + culture day 15 cell density) The Z2X (15-13) day specific production rate was calculated by dividing the concentration of the substance accumulated in the culture supernatant (mgZL) at the end of the culture by the cumulative cell density (cell ZmLZ day).
[0122] コウジ酸を ImmolZLの濃度で添加した場合の生細胞密度の経時変化を図 8に示 した。到達最大細胞密度は、コントロール培養では培養 11日目に 2. 9 X 106細胞/ 日であったのに対し、コウジ酸を添加した培養では培養 9日目に 2. 0〜3. 4 X 106細 胞 Z日であった。また、当該培養における細胞の生存率は、培養開始から培養 9日 目まで 80%以上の良好な値を示し、 15日目にはコントロール培養では 53%であつ たのに対し、コウジ酸を添カ卩した培養では 29〜57%であった。 [0122] Figure 8 shows the time course of the density of viable cells when kojic acid was added at a concentration of ImmolZL. The maximum cell density reached was 2.9 X 10 6 cells / day on day 11 in the control culture, whereas 2.0 to 3.4 X on day 9 in the culture supplemented with kojic acid. 10 6 cells Z days. In addition, the cell viability in the culture showed a good value of 80% or more from the start of the culture to the 9th day of culture, and on the 15th day it was 53% in the control culture, whereas kojic acid was added. In cultivated cultures, it was 29-57%.
[0123] 算出された、抗体の比生産速度をコウジ酸添加濃度および添加時期毎にそれぞれ 図 9に示した。培養開始と同時または 3日目にコウジ酸を添加した培養では、添加し たコウジ酸の濃度依存的に抗体の比生産速度は向上し、抗体の生産性向上効果は コントロールと比較して顕著であった。一方、培養開始から 5日目にコウジ酸を添加し た場合には、抗体の生産性向上効果は顕著ではなぐ添加したコウジ酸の濃度にも 比例していな力つた。  [0123] The calculated specific production rate of the antibody is shown in Fig. 9 for each kojic acid addition concentration and each addition time. In the culture in which kojic acid was added at the same time as the start of culture or on the third day, the specific production rate of the antibody increased depending on the concentration of the added kojic acid, and the antibody productivity was significantly improved compared to the control. there were. On the other hand, when kojic acid was added on the 5th day from the start of the culture, the effect of improving antibody productivity was not significant, and was not proportional to the concentration of added kojic acid.
[0124] 抗体の比生産速度は、 1. OmmolZLの濃度で培養開始から 3日目にコウジ酸を 添加した場合が最大になり、コウジ酸の添カ卩による抗体の生産性向上効果が確認さ れた。し力しながら、抗体の生産性向上効果が最大となった、この培養条件では、図 8に示されるように培養期間中における生細胞密度は、他の培養条件と比較して減 少していた。また、培養開始から 15日目での細胞の生存率は 27%と最も低いもので めつに。  [0124] The specific antibody production rate is as follows: 1. When kojic acid was added on the 3rd day from the start of culture at a concentration of OmmolZL, the effect of improving the antibody productivity by the addition of kojic acid was confirmed. It was. However, in this culture condition where the antibody productivity improvement effect was maximized, the viable cell density during the culture period decreased compared to the other culture conditions as shown in FIG. . In addition, the cell viability on the 15th day after the start of culture was the lowest at 27%.
[0125] 以上のことから、 709 LCA— 500G株の三角フラスコを用いた培養において、コゥ ジ酸を添加することにより抗体の生産性が向上することが確認された。し力しながら、 添加するコウジ酸の濃度および添加する時期は、生産に用いる細胞の増殖と比生産 速度とを考慮して設定する必要があることが明らかとなった。  [0125] From the above, it was confirmed that the antibody productivity was improved by adding kojic acid in the culture using the 709 LCA-500G Erlenmeyer flask. However, it has become clear that the concentration of kojic acid to be added and the timing of addition need to be set in consideration of the growth of cells used for production and the specific production rate.
また、 15日目の培養の終了時点に培養上清に蓄積された物質の濃度 (以下、累積 生産抗体濃度と記す)を、表 1に示した。併せて、コントロール培養に対する累積生産 抗体量の割合も示した。 Table 1 shows the concentrations of substances accumulated in the culture supernatant at the end of the 15th day of culture (hereinafter referred to as cumulative production antibody concentration). In addition, cumulative production for control culture The ratio of antibody amount is also shown.
[0126] 上記の通り、コウジ酸を培地に添加することで、細胞あたりの抗体の生産性は向上 するが、生産細胞の増殖は抑制される。しかしながら、表 1に示されるように、上記の いずれの条件でコウジ酸を添加した場合にも、培養終了時の累積生産抗体量は、コ ントロール培養と比較して上昇していた。即ち、コウジ酸を添加した培地で培養する 場合、物質の生産においては負の要因となる細胞増殖の抑制効果以上に、細胞あ たりの抗体の生産性を向上させる効果が高いことが確認された。  [0126] As described above, by adding kojic acid to the medium, the productivity of the antibody per cell is improved, but the growth of the production cell is suppressed. However, as shown in Table 1, even when kojic acid was added under any of the conditions described above, the amount of cumulatively produced antibody at the end of the culture increased compared to the control culture. That is, when cultured in a medium supplemented with kojic acid, it was confirmed that the effect of improving antibody productivity per cell was higher than the inhibitory effect of cell growth, which is a negative factor in the production of substances. .
産業上の利用可能性  Industrial applicability
[0127] 本発明を用いることにより、動物細胞を培養し、該細胞力 物質を生産させる方法 にお 、て、生産される物質の生産性を向上することができる。 [0127] By using the present invention, productivity of a substance to be produced can be improved in a method for culturing animal cells and producing the cellular force substance.

Claims

請求の範囲 The scope of the claims
[1] 以下の（a)〜（e)から選ばれる少なくとも 1つの物質を添加した培地中で、物質を生 産する能力を有する動物細胞を培養し、培養物中に該物質を生成蓄積させ、該培養 物から該物質を採取すること、を特徴とする物質の製造方法。  [1] Animal cells having the ability to produce a substance are cultured in a medium to which at least one substance selected from the following (a) to (e) is added, and the substance is produced and accumulated in the culture. A method for producing a substance, which comprises collecting the substance from the culture.
(a)リグナン類  (a) Lignans
(b)フラボノイド類  (b) Flavonoids
(c)ヒストン脱ァセチルイ匕酵素阻害剤  (c) Histone deacetylase inhibitor
(d)テルぺノイド類  (d) Terpenoids
(e)コウジ酸またはその誘導体  (e) Kojic acid or its derivatives
[2] テルぺノイド類がカンファである、請求項 1に記載の方法。  [2] The method according to claim 1, wherein the terpenoid is camphor.
[3] 動物細胞が哺乳類に属する動物細胞である、請求項 1または 2に記載の方法。  [3] The method according to claim 1 or 2, wherein the animal cell is an animal cell belonging to a mammal.
[4] 哺乳類に属する動物が霊長類または齧歯類である、請求項 3に記載の方法。  [4] The method according to claim 3, wherein the animal belonging to the mammal is a primate or a rodent.
[5] 動物細胞がミエローマ細胞または卵巣細胞、あるいはこれらの細胞に由来する細胞 である、請求項 1〜4のいずれか 1項に記載の方法。  [5] The method according to any one of claims 1 to 4, wherein the animal cell is a myeloma cell or ovary cell, or a cell derived from these cells.
[6] 物質がペプチドである、請求項 1〜5のいずれか 1項に記載の方法。 [6] The method according to any one of claims 1 to 5, wherein the substance is a peptide.
[7] 動物細胞がペプチドをコードする遺伝子を含有するベクターが導入された形質転換 細胞である、請求項 6に記載の方法。 7. The method according to claim 6, wherein the animal cell is a transformed cell into which a vector containing a gene encoding a peptide has been introduced.
[8] ペプチドが糖蛋白質である、請求項 6または 7に記載の方法。 [8] The method according to claim 6 or 7, wherein the peptide is a glycoprotein.
[9] 糖蛋白質が抗体である、請求項 8に記載の方法。 [9] The method of claim 8, wherein the glycoprotein is an antibody.
[10] 以下の（a)〜（e)から選ばれる少なくとも 1つの物質を添加した培地中で、物質を生 産する能力を有する動物細胞を培養し、該動物細胞より生産される物質の細胞あた りの生産性を向上させた後、培養物中に該物質を生成蓄積させ、該培養物から該物 質を採取することを特徴とする物質の製造方法。  [10] An animal cell having the ability to produce a substance is cultured in a medium to which at least one substance selected from the following (a) to (e) is added, and the substance cell produced from the animal cell is cultured. A method for producing a substance, comprising improving the productivity per production, producing and accumulating the substance in a culture, and collecting the substance from the culture.
(a)リグナン類  (a) Lignans
(b)フラボノイド類  (b) Flavonoids
(c)ヒストン脱ァセチルイ匕酵素阻害剤  (c) Histone deacetylase inhibitor
(d)テルぺノイド類  (d) Terpenoids
(e)コウジ酸またはその誘導体 (e) Kojic acid or its derivatives
[11] テルぺノイド類がカンファである、請求項 10に記載の方法。 [11] The method according to claim 10, wherein the terpenoid is camphor.
[12] 動物細胞が哺乳類に属する動物細胞である、請求項 10または 11に記載の方法。 12. The method according to claim 10 or 11, wherein the animal cell is an animal cell belonging to a mammal.
[13] 哺乳類に属する動物が霊長類または齧歯類である、請求項 12に記載の方法。 [13] The method according to claim 12, wherein the animal belonging to the mammal is a primate or a rodent.
[14] 動物細胞がミエローマ細胞または卵巣細胞、あるいはこれらの細胞に由来する細胞 である、請求項 10〜 13のいずれか 1項に記載の方法。 [14] The method according to any one of claims 10 to 13, wherein the animal cell is a myeloma cell or an ovary cell, or a cell derived from these cells.
[15] 物質がペプチドである、請求項 10〜14のいずれか 1項に記載の方法。 [15] The method according to any one of claims 10 to 14, wherein the substance is a peptide.
[16] 動物細胞がペプチドをコードする遺伝子を含有するベクターが導入された形質転換 細胞である、請求項 15に記載の方法。 16. The method according to claim 15, wherein the animal cell is a transformed cell into which a vector containing a gene encoding a peptide has been introduced.
[17] ペプチドが糖蛋白質である、請求項 15または 16に記載の方法。 17. The method according to claim 15 or 16, wherein the peptide is a glycoprotein.
[18] 糖蛋白質が抗体である、請求項 17に記載の方法。 18. The method according to claim 17, wherein the glycoprotein is an antibody.
[19] 以下の（a)〜（e)から選ばれる少なくとも 1つの物質を添加した培地中で、物質を生 産する能力を有する動物細胞を培養することを特徴とする、該動物細胞より生産され る物質の細胞あたりの生産性を向上させる方法。  [19] Produced from animal cells characterized by culturing animal cells having the ability to produce substances in a medium to which at least one substance selected from the following (a) to (e) is added: A method for improving the per-cell productivity of the substance.
(a)リグナン類  (a) Lignans
(b)フラボノイド類  (b) Flavonoids
(c)ヒストン脱ァセチルイ匕酵素阻害剤  (c) Histone deacetylase inhibitor
(d)テルぺノイド類  (d) Terpenoids
(e)コウジ酸またはその誘導体  (e) Kojic acid or its derivatives
[20] テルぺノイド類がカンファである、請求項 19に記載の方法。  [20] The method according to claim 19, wherein the terpenoid is camphor.
[21] 動物細胞が哺乳類に属する動物細胞である、請求項 19または 20に記載の方法。 21. The method according to claim 19 or 20, wherein the animal cell is an animal cell belonging to a mammal.
[22] 哺乳類に属する動物が霊長類または齧歯類である、請求項 21に記載の方法。 [22] The method according to claim 21, wherein the animal belonging to the mammal is a primate or a rodent.
[23] 動物細胞がミエローマ細胞または卵巣細胞、あるいはこれらの細胞に由来する細胞 である、請求項 19〜22のいずれか 1項に記載の方法。 [23] The method according to any one of claims 19 to 22, wherein the animal cell is a myeloma cell or an ovary cell, or a cell derived from these cells.
[24] 物質がペプチドである、請求項 19〜23のいずれか 1項に記載の方法。 [24] The method according to any one of claims 19 to 23, wherein the substance is a peptide.
[25] 動物細胞がペプチドをコードする遺伝子を含有するベクターが導入された形質転換 細胞である、請求項 24に記載の方法。 25. The method according to claim 24, wherein the animal cell is a transformed cell into which a vector containing a gene encoding a peptide has been introduced.
[26] ペプチドが糖蛋白質である、請求項 24または 25に記載の方法。 26. The method according to claim 24 or 25, wherein the peptide is a glycoprotein.
[27] 糖蛋白質が抗体である、請求項 26に記載の方法。 [27] The method of claim 26, wherein the glycoprotein is an antibody.
PCT/JP2006/321082 2005-10-24 2006-10-23 Method for production of substance WO2007049567A1 (en)

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