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WO2007049567A1 - Procede de production d’une substance - Google Patents

Procede de production d’une 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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WIPO (PCT)
Prior art keywords
cell
culture
cells
substance
antibody
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PCT/JP2006/321082
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English (en)
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.
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Application filed by Kyowa Hakko Kogyo Co., Ltd. filed Critical Kyowa Hakko Kogyo Co., Ltd.
Publication of WO2007049567A1 publication Critical patent/WO2007049567A1/fr

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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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  • Virology (AREA)
  • Biomedical Technology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne un procédé de production d’une substance, lequel est caractérisé par la culture d’une cellule animale dans un milieu de culture complété par au moins une substance choisie parmi une lignane, un flavonoïde, un inhibiteur d’histone désacétylase, un terpénoïde et l’acide kojique ou un dérivé de celui-ci. La présente invention concerne également un procédé pour améliorer la productivité d’une substance, lequel est caractérisé par la culture d’une cellule animale dans un milieu de culture complété par au moins une substance choisie parmi une lignane, un flavonoïde, un inhibiteur d’histone désacétylase, un terpénoïde et l’acide kojique ou un dérivé de celui-ci.
PCT/JP2006/321082 2005-10-24 2006-10-23 Procede de production d’une substance WO2007049567A1 (fr)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
JP2005-308943 2005-10-24
JP2005308943 2005-10-24
JP2006-043695 2006-02-21
JP2006043695 2006-02-21
JP2006118837 2006-04-24
JP2006-118837 2006-04-24

Publications (1)

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WO2007049567A1 true WO2007049567A1 (fr) 2007-05-03

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WO (1) WO2007049567A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2019503675A (ja) * 2015-12-16 2019-02-14 エフ．ホフマン−ラ ロシュ アーゲーＦ． Ｈｏｆｆｍａｎｎ−Ｌａ Ｒｏｃｈｅ Ａｋｔｉｅｎｇｅｓｅｌｌｓｃｈａｆｔ 改善された組換え体作製方法
CN116496967A (zh) * 2023-01-28 2023-07-28 广西中医药大学 一种提高微藻活性物质含量的促进剂及其应用

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JPS57170186A (en) * 1981-03-18 1982-10-20 Max Planck Gesellschaft Perfectly synthetic cell culture medium
JPH03292892A (ja) * 1990-04-12 1991-12-24 Idemitsu Petrochem Co Ltd 動物細胞による脂肪酸の製造法
JPH089968A (ja) * 1994-06-28 1996-01-16 Tosoh Corp ｎ−酪酸を含有することを特徴とする動物細胞培養用培地及び培養方法
JP2003144151A (ja) * 2001-11-09 2003-05-20 Kazutaka Miyatake ハイブリドーマ培養用培地及びモノクローナル抗体の生産方法
WO2004011644A1 (fr) * 2002-07-30 2004-02-05 Riken Methode de promotion d'une recombinaison homologue de cellules somatiques et methode d'elaboration d'un anticorps specifique
JP2005052004A (ja) * 2003-08-01 2005-03-03 Institute Of Physical & Chemical Research 気分安定薬の評価方法およびスクリーニング方法
JP2005521401A (ja) * 2002-03-27 2005-07-21 イミュネックス・コーポレーション ポリペプチド産生を増加させる方法

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS57170186A (en) * 1981-03-18 1982-10-20 Max Planck Gesellschaft Perfectly synthetic cell culture medium
JPH03292892A (ja) * 1990-04-12 1991-12-24 Idemitsu Petrochem Co Ltd 動物細胞による脂肪酸の製造法
JPH089968A (ja) * 1994-06-28 1996-01-16 Tosoh Corp ｎ−酪酸を含有することを特徴とする動物細胞培養用培地及び培養方法
JP2003144151A (ja) * 2001-11-09 2003-05-20 Kazutaka Miyatake ハイブリドーマ培養用培地及びモノクローナル抗体の生産方法
JP2005521401A (ja) * 2002-03-27 2005-07-21 イミュネックス・コーポレーション ポリペプチド産生を増加させる方法
WO2004011644A1 (fr) * 2002-07-30 2004-02-05 Riken Methode de promotion d'une recombinaison homologue de cellules somatiques et methode d'elaboration d'un anticorps specifique
JP2005052004A (ja) * 2003-08-01 2005-03-03 Institute Of Physical & Chemical Research 気分安定薬の評価方法およびスクリーニング方法

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2019503675A (ja) * 2015-12-16 2019-02-14 エフ．ホフマン−ラ ロシュ アーゲーＦ． Ｈｏｆｆｍａｎｎ−Ｌａ Ｒｏｃｈｅ Ａｋｔｉｅｎｇｅｓｅｌｌｓｃｈａｆｔ 改善された組換え体作製方法
CN116496967A (zh) * 2023-01-28 2023-07-28 广西中医药大学 一种提高微藻活性物质含量的促进剂及其应用
CN116496967B (zh) * 2023-01-28 2024-03-01 广西中医药大学 一种提高微藻活性物质含量的促进剂及其应用

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