WO2008069244A1 - Process for producing glycoprotein composition - Google Patents

Process for producing glycoprotein composition Download PDF

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Publication number
WO2008069244A1
WO2008069244A1 PCT/JP2007/073514 JP2007073514W WO2008069244A1 WO 2008069244 A1 WO2008069244 A1 WO 2008069244A1 JP 2007073514 W JP2007073514 W JP 2007073514W WO 2008069244 A1 WO2008069244 A1 WO 2008069244A1
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Prior art keywords
cells
sialic acid
cell
culture
glycoprotein
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PCT/JP2007/073514
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French (fr)
Japanese (ja)
Inventor
Yoshinobu Konno
Kentaro Sakai
Ryuma Nagano
Masamichi Koike
Toshiyuki Suzawa
Shinji Hosoi
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Kyowa Hakko Kogyo Co., Ltd.
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Publication of WO2008069244A1 publication Critical patent/WO2008069244A1/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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/34—Sugars

Definitions

  • the present invention relates to a method for producing a glycoprotein composition by culturing cells in a medium to which at least one substance selected from sialic acid, sialic acid polymer and oligosaccharide containing sialic acid is added, A method for culturing cells that produce a glycoprotein composition, a method for increasing the amount of sialic acid added to the glycoprotein composition produced from the cells, and a glycoprotein composition produced from the cells.
  • the present invention relates to a method for suppressing the addition amount of N-glycolylneuraminic acid.
  • Sialidase one of the enzymes involved in sugar chain catabolism and differentiation, is distributed in various tissues such as viruses, bacteria, protozoa, and other vertebrates.
  • Sialidase is an enzyme that removes sialic acid from the non-reducing terminus of glycoprotein sugar chains, and is a type of exo-type ⁇ -glycosidase that catalyzes the initial reaction of glycolysis.
  • animal endogenous sialidase regulates catabolism of biomolecules that have been glycosylated by controlling the initial reaction of glycosylation. It has become clear that it controls many important cell functions, such as affecting the immune mechanism (Non-patent document 2, Non-patent document 3).
  • the method for producing glycoprotein includes a step of obtaining a culture solution of glycoprotein and a step of purifying the glycoprotein from the culture solution.
  • the sugar chain structure of glycoprotein secreted from the production cell into the culture medium varies depending on the sugar chain modification mechanism of the production cell, but in many cases, the non-reducing terminal side of the sugar chain structure of the glycoprotein is It is known to be modified by a sialic acid residue having a negative charge (Non-patent document 2, Non-patent document 3). Since sialidase contained in cells producing glycoprotein leaks and accumulates in the culture medium from the cytoplasm of the production cells (Non-patent Document 4), it acts on the glycoprotein accumulated in the culture medium, resulting in sugar The sialic acid is eliminated from the sugar chain bound to the protein (Non-patent Document 5). Similarly, in the culture process in the production of glycoprotein, it has become clear that sialidase can desorb sialic acid from the sugar chain bound to the glycoprotein (Non-Patent Document 6). ).
  • Non-patent Document 7 The glycoprotein structure of the glycoprotein from which sialic acid has been removed becomes a structure in which the non-reducing end side of the non-reducing end is exposed, and the structure is localized in the liver, etc. Since it is captured and decomposed by the galactose receptor), the blood half-life of glycoprotein is greatly reduced by the elimination of sialic acid (Non-patent Document 8).
  • glycoprotein pharmaceuticals in order to obtain a glycoprotein in which sialic acid is added to the non-reducing end of the glycoprotein sugar chain, it is necessary to suppress the activity of sialidase leaked from production cells. Protein is considered important in the development of pharmaceuticals.
  • Patent Document 3 a method for producing a glycoprotein in which a sialidase inhibitor is added to a medium has been known.
  • N-acetylneuraminic acid which is a kind of sialic acid, and derivatives thereof are known (Non-patent Document 9).
  • N-acetylmethylneuraminic acid 2,3 dehydro-2 deoxy N-acetylmethylneuraminic acid (5-acetamido-2,6
  • draw 3,5 dideoxy D glycero D galactonone dienoic acid also referred to as Neu5A c2en or 2, 3 D
  • Neu5A c2en or 2 also inhibits CHO cell-derived sialidase
  • sialidase inhibitors become acidic when dissolved in the medium, some sialidase inhibitors cause growth inhibition, are not effective unless they are added properly, and osmotic pressure increases.
  • sialidase inhibitors are expensive, there are practical problems such as high costs when adding high concentrations of sialidase inhibitors to the medium.
  • Non-patent Document 12 a method is known in which sialic acid is added to the terminal side of a sugar chain of a glycoprotein by sialyl transferase.
  • sialyl transferase ⁇ 2 ⁇ 3 sialyltransferase (Patent Document 5), a 2 ⁇ 8 sialyltransferase (Patent Document 6) and the like are known.
  • the type of glycoprotein sugar chain is changed by changing the sugar composition or sugar concentration in the medium in cell culture.
  • Method Patent Document 7
  • Patent Document 8 a method of modifying the sugar chain structure by controlling the specific consumption rate of sugar in the medium
  • Non-patent Document 13 A method of increasing the sialic acid content in glycoproteins by culturing cells in a cetylmannosamine-added medium (Non-patent Document 13), and adding darcosamine or N-acetyldarcosamine to the cell culture medium.
  • Patent Document 9 A method for suppressing the transfer of galactose residues to the chain (Patent Document 9), adding alkanoic acid or its salt to the cell culture medium, and controlling the osmotic pressure or temperature to control the sialic acid of the glycoprotein sugar chain Including How to improve (Patent Document 10), glycoprotein using knockout cells were Shiaridaze gene Quality monitoring (Patent Document 11), monitoring CO concentration in cell culture media
  • the amount of N-glycolylneuraminic acid in the medium is controlled.
  • Patent Document 12 How to control (Patent Document 12) is known!
  • N-glycolylneuraminic acid or N-acetylneuraminic acid is mainly present as sialic acid (Non-patent Document 14).
  • N-glycolinorenolemic acid does not exist because of the lack of the conversion enzyme from CMP acetylenylamic acid to CMP glycolylneuraminic acid! / (Non-patent Document 15).
  • Non-patent Document 16 Non-patent Document 16
  • sugar chains containing N-glycolylneuraminic acid may be recognized as foreign sugar chains by the immune system and antibody production may occur. Therefore, when administering a glycoprotein composition to humans, it is preferable that N-glycolylneuraminic acid is not mixed.
  • Patent Document 1 W096 / 39488
  • Patent Document 2 US4724206
  • Patent Document 3 US5510261
  • Patent Document 4 US6528286
  • Patent Document 5 Japanese Patent No. 3131322
  • Patent Document 6 Japanese Patent No. 3394256
  • Patent Document 7 JP-A-6-292592
  • Patent Document 8 WO02 / 02793
  • Patent Document 9 Japanese Patent Laid-Open No. 11-127890
  • Patent Document 10 US5705364
  • Patent Document 11 EP0700443
  • Patent Document 12 W095 / 12684
  • Non-patent literature 1 Nature Rev. Drug. Discov., 3, 383-384 (2004)
  • Non-patent literature 2 Trends Biochem. Sci., 10, 357-360 (1985)
  • Non-patent literature 3 Glycobiology, 3, 201 (1993)
  • Non-Patent Document 4 Glycobiology, 3, 455 (1993)
  • Non-Patent Document 5 Bio / Technology, 13, 692 (1995)
  • Non-Patent Document 6 Biotechnology progress, 20, 864 (2004)
  • Non-Patent Document 7 Journal of Biological Chemistry, 265, 12127 (1990)
  • Non-Patent Document 8 Blood, 73, 84 (1998)
  • Non-Patent Document 9 Handbook of Enzyme Inhibitors ard, revised and enlar ged edition Part A, Wiley— VCH, Weinheim (1999)
  • Non-Patent Document 10 Biotech. Bioeng., 55, 390 (1997)
  • Non-Patent Document 11 Biotechnology, 13, 692 (1995)
  • Non-Patent Document 12 J. Am. Chem. Soc., 108, 2068 (1986)
  • Non-Patent Document 13 Biotech. Bioeng., 58, 642 (1998)
  • Non-Patent Document 14 Nature Medicine, 11, 228 (2005)
  • Non-Patent Document 15 Proc. Natl. Acad. Sci., USA 100, 12045 (1998)
  • Non-Patent Document 16 FEBS Lett., 275, 9 (1990)
  • An object of the present invention is to provide a method for producing a glycoprotein composition having an increased amount of sialic acid added to a sugar chain, and to provide a glycoprotein composition having an improved amount of sialic acid added to a sugar chain.
  • To provide a method for culturing cells to be produced to provide a method for improving the amount of sialic acid added to a sugar chain in a glycoprotein composition, and to suppress the amount of ⁇ ⁇ ⁇ -glycolylneuraminic acid in a glycoprotein composition It is to provide a method.
  • the present invention relates to the following (1) to (32).
  • a method for producing a glycoprotein composition comprising culturing cells in an added medium, producing and accumulating the glycoprotein composition in the culture, and collecting the glycoprotein composition from the culture. .
  • At least one substance selected from sialic acid, sialic acid polymer and oligosaccharide containing sialic acid is added to the medium at the start of culture, logarithmic growth phase, or stationary phase, (1) or (2 ) Method.
  • Herbal HEK293 cells Herbal HEK293 cells.
  • Cell force The method according to any one of (1) to (6), wherein the cell is a cell into which a DNA encoding a glycoprotein has been introduced.
  • At least one substance selected from sialic acid, sialic acid polymer and oligosaccharide containing sialic acid is added to the medium at the start of culture, logarithmic growth phase, or stationary phase, (9) or ( The method according to 10).
  • Herbal HEK293 cells Herbal HEK293 cells.
  • At least one substance selected from sialic acid, sialic acid polymer and oligosaccharide containing sialic acid is added to the medium at the start of culture, logarithmic growth phase, or stationary phase, (17) or ( The method described in 18).
  • the cell is cultured at an osmotic pressure of 250 to 400 mOsm / kg, any of (17) to (; 19)
  • Herbal HEK293 cells Herbal HEK293 cells.
  • At least one substance selected from sialic acid, sialic acid polymer and oligosaccharide containing sialic acid is added to the medium at the start of culture, logarithmic growth phase, or stationary phase, (25) or ( The method described in 26).
  • the cell is cultured at an osmotic pressure of 250 to 400 mOsm / kg, any of (25) to (27)
  • Herbal HEK293 cells (31) Cell force The method according to any one of (25) to (30), wherein the cell is a cell into which DNA encoding a glycoprotein has been introduced.
  • a glycoprotein composition characterized by culturing animal cells in a medium to which at least one substance selected from sialic acid, a sialic acid polymer and an oligosaccharide containing sialic acid is added.
  • Production method, culture method for glycoprotein-producing cells, method for improving the amount of sialic acid added to the sugar chain in the glycoprotein composition, and suppression of the amount of N-glycolylneuraminic acid in the glycoprotein composition A method is provided.
  • the glycoprotein produced according to the present invention increases the amount of sialic acid added to a glycoprotein produced by culturing without adding sialic acid, a sialic acid polymer or an oligosaccharide containing sialic acid. -Glycolinole neurolamic acid content is reduced and is useful as a medicine.
  • FIG. 1 Cultivation of Fedbachii culture using antithrombin-producing CHO cell line and medium containing various concentrations of N-acetylneuraminic acid in Erlenmeyer flasks Day 4 The relative sialic acid addition rate in is shown.
  • FIG. 2 shows the viable cell density and viability when an antithrombin-producing CHO cell line was used and cultured in a Erlenmeyer flask and fed-batch culture using a medium supplemented with N-acetylneuraminic acid. Viable cell density is indicated by a solid line and viability is indicated by a dotted line.
  • ⁇ is a control group without addition of N-acetylneuraminic acid
  • ⁇ is a group with addition of 20 mmol / L of N-acetylneuraminic acid
  • ⁇ is a group with addition of 40 mmol / L of N-acetylneuraminic acid
  • Laminic acid 6 Ommol / L added group mouth shows N-acetylethylneuraminic acid 80 mmol / L added group
  • country shows N-acetylethylneuraminic acid 100 mmol / L added group.
  • FIG. 3 Cumulative cell density versus protein production concentration when anti-thrombin-producing CHO cell line was used and fedbatch culture was performed in a Erlenmeyer flask supplemented with N-acetylneuraminic acid.
  • ⁇ is a control group without addition of N-acetylneuraminic acid
  • ⁇ is a group with addition of 20 mmol / L of N-acetylneuraminic acid
  • ⁇ addition of 40 mmol / L of N-acetylneuraminic acid Group
  • ⁇ is a group added with 60 mmol / L of N-acetylneuraminic acid
  • mouth is a group added with 80 mmol / L of N-acetylethylneuraminic acid
  • country is a group added with 100 mmol / L of N-acetylethylneuraminic acid. Show.
  • FIG. 4 shows the relative sialic acid addition rate on the 14th day of culture when Fedbach culturing was performed using antithrombin-producing CHO cell line and medium supplemented with N-acetylneuraminic acid in an Erlenmeyer flask.
  • FIG. 5 shows the relative sialic acid addition rate on the 14th day of culture when Fuedbatch culture was performed using antithrombin-producing CHO cell line and medium supplemented with sialic acid dimer in an Erlenmeyer flask.
  • FIG. 6 shows the relative sialic acid addition rate on the 14th day of culture when fed-batch culture was performed using antithrombin-producing CHO cell line and medium containing colominic acid in an Erlenmeyer flask.
  • FIG. 7 shows the relative sialic acid addition rate on the 14th day of culture when Fedbachii culture was performed using antithrombin-producing CHO cell line and medium containing oligosaccharide containing sialic acid in an Erlenmeyer flask.
  • the mouth indicates the N-acetylylneuraminic acid addition group
  • the ⁇ indicates the ⁇ 2,3-sialyllatatose addition group
  • the ⁇ indicates the ⁇ 2,6-sialyllatatose addition group.
  • FIG. 8 shows the relative sialic acid addition rate according to the timing of N-acetylneuraminic acid addition when fed batch culture was performed in an Erlenmeyer flask using an antithrombin-producing CHO cell line.
  • FIG. 9 shows the viable cell density and viability depending on the timing of N-acetylneuraminic acid addition when fed batch culture was performed in an Erlenmeyer flask using an antithrombin-producing CHO cell line.
  • ⁇ is a control group without addition of N-acetylneuraminic acid
  • ⁇ is a group with addition of N-acetylneuraminic acid on the 5th day
  • ⁇ is a group with addition of N-acetylethylneuraminic acid on the 8th day
  • ⁇ is N-acetylethylneuraminine Acid 9-day addition group
  • mouth is N-acetylneuraminic acid 10-day addition group
  • country is N-acetylneuraminic acid 11-day addition group
  • ⁇ is N-acetylneuraminic acid 12-day addition group
  • ⁇ N-acetylethyl neuraminic acid added on day 13 respectively.
  • FIG. 11 Relative content of N-glycolylneuraminic acid when fed-batch culture using antithrombin-producing CHO cell line and medium containing various concentrations of N-acetylneuraminic acid in a bioreactor Indicates.
  • the content of N-glycolylneuraminic acid in the sialic acid on the 8th day after N-acetylethylneuraminic acid-free culture is shown as 100%.
  • the mouth indicates the N-acetyl-neuraminic acid-free control group
  • the country indicates the 20-mmol / L N-acetyl-neuraminic acid-added group
  • the ⁇ indicates the 40-mmol / L N-acetyl-neuraminic acid-added group.
  • FIG. 12 Relative sialic acid on day 14 of culture when fed batch culture using antithrombin-producing CHO cell line and using bioreactor with medium containing various concentrations of N-acetylneuraminic acid was performed. Indicates the rate of addition.
  • the hatched lines indicate the N-acetyl-neuraminic acid-free control group, the country indicates the 20-mmol / L N-acetyl-neuraminic acid-added group, and the mouth indicates the 40-mmol / L-N-acetyl-neuraminic acid-added group.
  • FIG. 13 shows the viable cell density and viability when fed-batch culture was carried out in a bioreactor using antithrombin-producing CHO cell line and medium supplemented with various concentrations of N-acetylneuraminic acid.
  • ⁇ indicates a control group without N-acetylneuraminic acid added
  • country indicates a group with 20 mmol / L N-acetylneuraminic acid added
  • mouth indicates a group with 40 mmol / L N-acetylneuraminic acid added.
  • cells are cultured in a medium to which at least one substance selected from sialic acid, a sialic acid polymer and an oligosaccharide containing sialic acid is added, and a glycoprotein composition is produced and accumulated in the culture.
  • the present invention relates to a method for producing a glycoprotein composition and a method for culturing a glycoprotein-producing cell, wherein the glycoprotein composition is collected from the culture.
  • sialic acid means 2-keto-3-deoxynonic acid having a carboxyl group consisting of nine carbon skeletons, and is also called neuroamic acid.
  • N-acetylenorelamic acid Nemamic acid
  • N-glycolylneuraminic acid Nemamic acid
  • KDN diamineneuraminic acid
  • sialic acid derivatives include those obtained by modifying the above sialic acid such as acetylation, lactylation, and sulfation.
  • N-acetylyl 4-O-neuraminic acid N-acetylyl 9--O neuraminic acid, N-acetylyl 8,9-di-O neuraminic acid, N-acetylenole 9 0-ratato norenolamic acid
  • N-acetylyl 4-O Acetyl 9-ratatoylneuraminic acid N-acetyl-neuraminic acid 9-phosphoric acid
  • N-glycolyl 9-O-acetylneuraminic acid N Daricoleunor 9 O ratatoinorenolamic acid, N-glycolenorenolamic acid 8-stone
  • 5-azidoneuraminic acid N-acetyl-9-acetamido-9-deoxyneuraminic acid, N-acetyl-9-azido 9, deoxyneuraminic acid or 5-azidoneuraminic acid.
  • the sialic acid polymer is a polymer obtained by bonding 2 to 8 or 2 to 9 in the above-described at least two molecules of sialic acid or sialic acid derivative S, usually ⁇ configuration.
  • Any one can be used, for example, colominic acid, acetylneuraminic acid oligomer, or a sodium salt thereof.
  • colominic acid having an average chain length of 16 units including 2 ⁇ 8 a binding units derived from E. coli K1 strain or 2 ⁇ 8 binding sialic acid units and 2 ⁇ 9 derived from Escherichia coli K92 strain
  • Examples thereof include colominic acid having a combined chain sialic acid unit and an average chain length of 78 units.
  • examples of oligosaccharides containing sialic acid include oligosaccharide-linked sialic acids in which the amide substituent at the 5-position of sialic acid is substituted with an oligosaccharide.
  • Specific examples include ⁇ 2,3 sialyl latatos, ⁇ 2,6 sialyl latatos, disialyl latatos or sialyl latatosamine.
  • sialic acid, sialic acid polymer or oligosaccharide containing sialic acid used in the present invention can be obtained by a known method such as production by chemical synthesis or genetic engineering techniques.
  • commercially available sialic acid, sialic acid polymer or oligosaccharide containing sialic acid can also be used.
  • sialic acid, sialic acid polymer or oligosaccharide containing sialic acid include colominic acid sodium salt, ⁇ acetylneuraminic acid, ⁇ glycolylneuraminic acid, ⁇ acetylneuraminic acid oligomer Examples thereof include sodium salt and ⁇ 2,6 sialyl ratatose (hereinafter, Malkin Bio).
  • the sialic acid or the like used in the present invention is added at a higher concentration than sialic acid or the like added to a medium known so far.
  • the concentration of sialic acid is 5 ⁇ 200mmol / L, preferably 10 to 150 mmol / L, more preferably 20 to 100 mmol / L.
  • Concentration of sialic acid polymer (from about 0.2 ;; to 200 mmol / L, preferably (from about 0.2 to 150 mmol / L, more preferably from about 0.5 to 100 mmol / L.
  • Oligosaccharides containing sialic acid The concentration of; is from !! to 200 mmol / L, preferably from 2 to 150 mmol / L, more preferably from 5 to 1 OOmmol / L.
  • sialic acid or the like may be added to the medium over a plurality of periods, such as at the start of culture, in the logarithmic growth phase, or in the stationary phase.
  • the logarithmic growth phase refers to a time when the number of cells increases logarithmically with time in cell culture.
  • the quiescent phase is a period before the period when the number of living cells increases after the logarithmic growth phase and starts to decrease with a decrease in the survival rate.
  • the glycoprotein composition is a composition containing a glycoprotein molecule having a sugar chain in which a sialic acid addition site is present at the non-reducing end, and is an N-glycoside-linked sugar chain or A composition comprising a glycoprotein molecule having an O-glycoside-linked sugar chain is used. Since glycoprotein molecules are linked to various sugar chains by the sugar chain control mechanism of the produced cells, the glycoprotein becomes a composition of glycoprotein molecules having these various sugar chain structures. That is, in the present invention, the glycoprotein composition is a glycoprotein molecule having a sugar chain having a sialic acid addition site at the non-reducing end and having an N-glycoside-bonded sugar chain or an O-glycoside-bonded sugar chain. Any other sugar chain structure may be used in the composition.
  • the cells in the present invention may be any cells as long as they are capable of producing a glycoprotein composition, and specifically include the ability to include yeast, insect cells, plant cells, or animal cells. S, preferably animal cells.
  • Examples of cells capable of producing a glycoprotein composition include transformed cells into which a vector containing a gene encoding a glycoprotein has been introduced.
  • a transformed cell into which a vector containing a gene encoding a glycoprotein has been introduced can be obtained, for example, by introducing a recombinant vector containing a DNA encoding a glycoprotein and a promoter into the host cell. wear.
  • yeast examples include microorganisms belonging to the genus Saccharomyces, Schizosaccharomyces, Kluybe mouth mouth, Genus Trichospolon or Schneomyces, such as Saccharomvces c erevisiae ⁇ Schizosaccharomyces pomoe ⁇ Kluyveromvc es lactis. With the power S to raise poron pullulans, Schwanniomyces alluvius or Pichia nastoris.
  • Insect cells include Spodoptera frugiperda's ovarian cells Sf 9, Sf21 [Current 'Protocorenoles' in' Molecular. Neurology; Baculovirus Expression Vectors, A Laboratory Manual, WH Freeman and company, New York (1992) High 5 (manufactured by Invitrogen), which is an ovary cell of spider or Trichoplusiani, can be mentioned.
  • plant cells it is possible to raise cells derived from tobacco, potatoes, tomatoes, carrots, soybeans, rapes, alfa norefas, rice, wheat, barley, green moss or duckweed with a force S.
  • animal cells include cells derived from rats, mice, humans, monkeys, dogs, hamsters, etc., or hybrid cell lines of these animal cells.
  • specific examples of animal cells include Chinese nomstar ovarian tissue-derived cell lines CHO, CHO-KKATC C CCL—61), CHO / dhfr— (ATCC CRL—9096), Pro5 strain (ATCC CRL—1781), CHO — S (Invitrogen Cat # 11619), mouse cell line NS0 (A TCC CRL—1827), SP2 / 0 (ATCC CRL—1581), mouse myeloma cell line S P2 / 0— Agl4, rat myeloma cell line Y3 Agl 2. 3.
  • any of the commonly used culture methods can be used.
  • batch culture repeat batch culture, fed-batch culture or perfusion culture.
  • the Fuedbachi culture is a culture method in which physiologically active substances, nutrient factors, and the like are additionally supplied in small amounts continuously or intermittently.
  • the fed-batch culture can prevent a decrease in the reached cell density of the cultured cells due to accumulation of waste products in the culture solution in which the metabolic efficiency of the cells is high.
  • the desired glycoprotein composition in the collected culture broth is at a higher concentration than that obtained in batch culture, the glycoprotein composition can be easily separated and purified, and compared to notch culture.
  • the production amount of the glycoprotein composition per medium volume can be increased.
  • sialic acid can be added to the solution added to the medium, it is easy to control the sialic acid concentration in the culture medium.
  • 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 fresh medium is newly supplied to the culture tank. Is the method. This method is preferable because the culture environment in the culture tank is always kept good.
  • any medium that contains a carbon source, nitrogen source, inorganic salts, and the like that can be assimilated by yeast, and that can efficiently culture transformants can be used.
  • Either a synthetic medium or a semi-synthetic medium supplemented with natural products for the purpose of supplementing the function of the synthetic medium may be used.
  • carbon sources include those that can be assimilated by yeast, such as glenolec, fructose or sucrose, or carbohydrates such as molasses, starch or starch hydrolysates, organic acids such as acetic acid or propionic acid.
  • alcohols such as ethanol or propanol can be used.
  • Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate or ammonium phosphate such as ammonium phosphate, other nitrogen-containing compounds, peptone, meat extract, yeast extract, corn steep liquor, strength Zein hydrolyzate, soybean meal or soybean meal hydrolyzate, various fermented bacterial cells, or digests thereof can be used.
  • the inorganic salt monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, or calcium carbonate can be used.
  • Yeast culture is usually performed under aerobic conditions such as shaking culture or deep aeration and agitation culture.
  • the culture temperature is preferably 15 to 40 ° C, and the culture time is usually 16 hours to 7 days.
  • Cultivation The pH during cultivation is usually maintained at 3 ⁇ 0 to 9 ⁇ 0.
  • ⁇ is prepared using inorganic or organic acid, alkali solution, urea, calcium carbonate, ammonia, etc.
  • antibiotics such as an ampicillin and tetracycline
  • cultivation may add an inducer to a culture medium as needed.
  • Common media used in insect cell culture are: ⁇ —FH medium (Betaton 'Dickinson'), Sf—900 II SFM medium (Invitrogen), EX—CELL TM 400, EX — CELL TM 405 [both manufactured by Sigma's Aldrich 'Fine Chemical (hereinafter referred to as SAFC)] or Grace's Insect Medium [Natur, 195, 788 (1962)] can be used.
  • SAFC Sigma's Aldrich 'Fine Chemical
  • Insect cells are usually cultured under conditions of pH 6 to 7, 25 to 30 ° C, etc .; for! To 5 days. Further, an antibiotic such as gentamicin may be added to the medium as needed during the culture.
  • MS Murashige & 'Sturg
  • White medium or a medium in which plant hormones such as auxin and cytokinin are added to these mediums, etc. Can be used.
  • Plant cells are usually cultured for 3 to 60 days under conditions of pH 5 to 9 and 20 to 40 ° C.
  • Any medium can be used as a medium used for culturing animal cells as long as it is a basal medium used for culturing ordinary animal cells.
  • RPMI1640 medium L The Journal oi the American Medical Association, 199, 519 (1967)], Eagle's MEM medium [Science, 122. 501 (1952)], Dulbecco's modified MEM medium [Virology, 8, 396 (1959)), 199 medium (Proceeding of the Society for the Biological Medicine, 73, 1 (1950)), F12 medium (Proc.
  • IMDM medium J. Experimental Medicine, 147, 923 (1978)]
  • DMEM medium Hybridoma Serum Free medium (Invitrogen), Chemically Defined Hybridoma Serum F Ree medium (Invitrogen), EX-CELL TM -302 medium (SAFC) or EX-CELL TM -CD-CHO (SAFC)
  • physiologically active substances or nutrient factors necessary for the growth of animal cells can be added as necessary. These additives can be added to the medium in advance before culturing and / or added to the medium during culturing.
  • an additional solution is appropriately added to the culture medium.
  • the additional supply method may be any form such as one solution or a mixed solution of two or more kinds, and the addition method may be continuous or intermittent.
  • physiologically active substance examples include insulin, IGF-1, transferrin, albumin, and coenzyme Q.
  • Nutritional factors include sugar, amino acids, vitamins and hydrolysates
  • lipid etc. are mention
  • sugar examples include glucose, mannose, and fructose, and they are used alone or in combination of two or more.
  • Amino acids include L-alanine, L-arginine, L-asparagin, L-aspartic acid, L-cystine, L--glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-fee Examples include ninorealanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine or L-valine, and are used alone or in combination of two or more.
  • Vitamins include d biotin, D pantothenic acid, choline, folic acid, myo inositol, niacinamide, pyridoxal, riboflavin, thiamine, cyanocobalamin or DL-a tocopherol, and one or a combination of two or more. Used.
  • the hydrolyzate include hydrolyzed soybeans, wheat, rice, peas, corn, cottonseed, yeast extract and the like.
  • lipids include cholesterol, linoleic acid, and linolenic acid.
  • antibiotics such as kanamycin, streptomycin, penicillin or hygromycin can be added to the medium as needed during culture.
  • dissolved oxygen concentration control pH control, temperature control, stirring, etc. can be performed according to the usual methods used for culturing various cells.
  • the pH should be adjusted to pH 5-9, preferably pH 6-8, which is a neutral range suitable for cell growth and substance production. Good.
  • the osmotic pressure suitable for cell culture and substance production is 200 to 600 mOsm / kg, preferably 250 to 500 mOsm / kg, more preferably 250 to 400 mOsm / kg.
  • the method for controlling the osmotic pressure in the medium in the present invention is a substance that adjusts the osmotic pressure in the medium.
  • a method of changing the concentration of the osmotic pressure regulator hereinafter referred to as an osmotic pressure regulator
  • a method of adding an osmotic pressure solution different from the culture medium a method of adding a solution in which the osmotic pressure regulator is dissolved, or a medium containing
  • the method of changing the molar concentration or electrolysis of the medium depending on the substance produced in (1) is a method of changing the concentration of the osmotic pressure regulator.
  • Examples of the method for changing the concentration of the osmotic pressure adjusting agent in the medium include a method of adding the osmotic pressure adjusting agent to the medium and a method of removing the osmotic pressure adjusting agent in the medium.
  • the osmotic pressure adjusting agent may be any substance that changes the osmotic pressure of the medium, that is, a substance that changes the molar concentration or the electrolysis degree. Specifically, sodium chloride, chloride may be used.
  • Salts such as potassium or lithium chloride, sugars such as gnolecose, galactose, mannose, fucose, funolectose, sucrose, mannitol, xylose, trehalose, sorbitol or glycerol, various amino acids, vitamins, soybeans, wheat, rice or corn Plant-derived hydrolysates, animal-derived proteins such as ushi protein hydrolysates, yeast extracts such as yeast ixstrata, alkalis used to adjust the pH of media such as sodium hydroxide, sodium carbonate or sodium bicarbonate Or coenzyme Q, creatine, ficoll, butyric acid Medium additives such as
  • sodium chloride is preferred.
  • Examples of the method of adding a solution having an osmotic pressure different from that of the medium to the medium include a method of adding a solution having a higher osmotic pressure, a solution or a lower osmotic pressure, and a solution.
  • Examples of high osmotic pressure solutions include solutions in which one or more salts such as sodium chloride are added to a medium that can be used for animal cells, and solutions that contain high concentrations of amino acids or vitamins. Or, a solution in which salts, amino acids, vitamins, etc. are added to a medium that can be used for animal cells.
  • As a low osmotic pressure solution sodium chloride is used from a medium usable for animal cells.
  • a solution from which one or more salts such as amino acids are removed a solution composed of a minimum of medium components such as amino acids and vitamins, a solution obtained by diluting a medium usable for animal cells with water, or water Etc.
  • the glycoprotein composition obtained by the production method of the present invention has a sialic acid per molecule as compared with a glycoprotein composition obtained by culturing cells without adding sialic acid or the like to the medium.
  • the added amount increases.
  • a glycoprotein composition having an increased amount of sialic acid per molecule means that sialic acid is bound to 50% or more, preferably 60% or more, more preferably 70% or more of the sialic acid addition site of the glycoprotein molecule.
  • This glycoprotein composition has improved physiological activity when administered in vivo as compared to a glycoprotein composition obtained without adding sialic acid or the like to the medium. Examples of the physiological activity include affinity with a receptor, stability in blood, pharmacological activity or immunogenicity.
  • the glycoprotein composition obtained by the method of the present invention is not particularly limited as long as it is a protein composition to which a sugar chain is bound, and preferred examples include eukaryotic cell-derived glycoprotein compositions. Preferred is a mammalian cell-derived glycoprotein composition, and more preferred is a human cell-derived glycoprotein composition. Specific examples include compositions comprising the following glycoproteins. Antibody, erythropoietin (EPO). Biol. Chem., 252.
  • TPO thrombopoietin
  • tissue-type plasminogen activator prolokinase
  • thrombomodulin anti Thrombin
  • ⁇ 1-antitrypsin C1 inhibitor
  • haptoglobin activated protein C
  • blood coagulation factor VII blood coagulation factor VIII
  • blood coagulation factor IX blood coagulation factor X
  • blood coagulation factor XI blood coagulation factor XII
  • blood Coagulation factor XIII prothrombin complex, fibrinogen, albumin, gonadotropin, thyroid stimulating hormone, epidermal growth factor (EGF), hepatocyte growth factor (HGF), keratinocyte growth factor, activin, osteogenic factor, granulocyte colony sting Intense Factor (G-CSF).
  • the antibody may be any antigen-binding antibody, such as an antibody that binds to a tumor-related antigen, an antibody that binds to an antigen related to allergy or inflammation, an antibody that binds to an antigen related to cardiovascular disease, self IgG is the preferred class of antibody that preferably binds to an antigen associated with an immune disease or an antibody that binds to an antigen associated with a viral or bacterial infection.
  • Anti-GD2 antibody [Anticancer Res., 13, 331-336 (1993)], JrLGD3irL body [Cancer Immunol. Immunother., 36 260-2 Ri., (1993)], GM2 antibody [Cancer Res., 54, 1511-1516, (1994)], anti-HER2 antibody [Proc. Natl.
  • Anti-interleukin 6 antibody [Immunol. Rev., 127, 5-24 (1992)], anti-interleukin 6 receptor antibody [Molecular Immunol. 31, 371-381 (1994)], anti-interleukin 5 antibody [Immunol. Rev., 1 27, 5-24 (1992)], anti-interleukin 5 receptor antibody, anti-interleukin 4 antibody [Cytokine, 3 , 562-567 (1991)], anti-interleukin 4 receptor antibody. Immunol.
  • Antibodies that bind to antigens associated with autoimmune diseases such as psoriasis, rheumatoid arthritis, Crohn's disease, ulcerative colitis, systemic lupus erythematosus, and multiple sclerosis include anti-self DNA antibodies [Immunol.
  • Antibodies that bind to antigens associated with viral or bacterial infections include anti-gpl20 antibodies [Structure, 8, 385-3 95 (2000)], anti-CD4 antibody Rheumatology, 25, 2065-2076, (199 8)], anti-CCR4 antibody, anti-verotoxin antibody Clin. Microbiol., 37, 396-399 (1999)], etc. .
  • an antibody includes an antibody fragment and a fusion protein containing an Fc region.
  • Antibodies include antibodies produced by gene recombination techniques, in addition to antibodies secreted by hybridoma cells prepared from spleen cells of immunized animals, and antigen expression in which the antibody gene is inserted. Examples thereof include antibodies obtained by introducing a vector into a host cell. Specific examples include antibodies produced by Hypridoma, human chimeric antibodies, humanized antibodies or human antibodies.
  • a hybridoma refers to a cell obtained by cell fusion of a B cell obtained by immunizing a mammal other than a human with an antigen and a myeloma cell derived from a rat or mouse.
  • the human chimeric antibody is a non-human animal antibody heavy chain variable region (hereinafter, the heavy chain is referred to as H chain, the variable region is also referred to as HV or VH) and an antibody light chain variable region (hereinafter referred to as "H chain”).
  • the light chain is also referred to as LV or VL as the 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! / )).
  • the non-human animal any animal such as a mouse, rat, hamster or rabbit can be used as long as it can produce a hyperidoma.
  • 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. Therefore, it is possible to construct a human chimeric antibody expression vector, introduce it into a host cell, express it and produce it.
  • the CH of the human chimeric antibody may be any CH as long as it belongs to human immunoglobulin (hereinafter referred to as “hlg”), but the hlgG class is preferred, and MgGl, hi gG2, MgG3 belonging to the hlgG class are preferred. Any of the subclasses such as MgG4 can be used.
  • the CL of the human chimeric antibody may be any as long as it belongs to hlg, and those of ⁇ class or ⁇ class can be used.
  • a human antibody is also referred to as a transplanted antibody (complementarity determining region: hereinafter referred to as CDR).
  • CDR complementarity determining region
  • Humanized antibodies are derived from VH and V of non-human animal antibodies. This refers to an antibody in which the amino acid sequence of the CDR of L is grafted to an appropriate position of human antibody VH and VL.
  • Humanized antibodies are constructed by constructing DNA encoding the V region by grafting the VH and VL CDR sequences of non-human animal antibodies to the VH and VL CDR sequences of any human antibody. It is possible to construct a humanized antibody expression vector by introducing it into an expression vector for a host cell having a gene encoding CL, and to express it by introducing the expression vector into a host cell.
  • the CH of the humanized antibody may be any as long as it belongs to hlg, but the MgG class is preferable, and any of the subclasses such as MgGl, MgG2, MgG3, or MgG4 belonging to the hlg G class can be used. it can.
  • the CL of the humanized antibody may be any as long as it belongs to hlg, and those of ⁇ class or e class can be used.
  • the vector that expresses the glycoprotein composition may be a vector or a chromosome that can replicate autonomously in the host cell. Those that contain a promoter that can be incorporated into the DNA and that can transcribe DNA encoding the target glycoprotein molecule are used.
  • yeast is used as a host cell
  • examples of the expression vector include YEP13 (ATCC 37115), YEp24 (ATC C 37051), YCp50 (ATCC 37419), and the like. Any promoter can be used as long as it can be expressed in a yeast strain.
  • Examples include promoters of cornose sugar genes such as hexose kinase, PH05 promoter 1, PGK promoter, GAP Promoter, ADH promoter, gal 1 promoter 1, gal 10 promoter, heat shock protein promoter, MF a 1 promoter or CUP 1 promoter.
  • any method can be used as long as it introduces DNA into yeast.
  • the Elect Mouth Position Method [Methods, in Enzymol., 194, 182 (19 90) ]
  • Spheroplast method [Proc. Natl. Acad. Sci. US A, 84, 1929 (1978)] or lithium acetate method. Bacteriology, 153, 163 (1983); Pro c. Natl. Acad. Sci. , 75, 1929 (1978)].
  • expression vectors such as pcDNAI and pcDM 8 (commercially available from Funakoshi), pAGE107 [JP-A-3-22979; Cytotechnology, 3, 133, (1990)], pAS3-3 (JP-A-2-227075), pCDM8 [Nature, 329, 840, (1987)] , PcDNAl / Amp (manufactured by Invitrogen), pREP4 (manufactured by Invitrogen), or PAGE103. Biochemistry, 101, 1307 (1987)]. Any promoter can be used as long as it can be expressed in animal cells.
  • cytomegalovirus (CMV) IE immediate ealy gene promoter
  • SV40 early promoter SV40 early promoter
  • retrovirus promoter SV40 early promoter
  • Meta mouth thionein promoter heat shock promoter or SRa promoter
  • An enhancer of the IE gene of human CMV may be used together with a promoter.
  • any method for introducing a recombinant vector into an animal cell any method can be used as long as it introduces DNA into an animal cell.
  • electroporation method [Cytotechnology, 3, 133 (1990)]
  • calcium phosphate Method Japanese Patent Laid-Open No. 2-227075
  • lipofusion method Proc. Natl. Acad. Sci.
  • insect cells When insect cells are used as the host, for example, Current 'Protocols' in Molecula I. Noroiroshi, Baculovirus Expression Vectors, A Laboratory Manu al, WH Freeman and Company, New York (1992) or Bio / Technology, 6, 47 (1988) and the like, the ability to express proteins can be achieved. Specifically, a recombinant gene transfer vector and a defective baculovirus genome are co-introduced into an insect cell to obtain a recombinant virus in the insect cell culture supernatant, and then the recombinant virus is further infected into the insect cell, and the protein is added.
  • a recombinant gene transfer vector and a defective baculovirus genome are co-introduced into an insect cell to obtain a recombinant virus in the insect cell culture supernatant, and then the recombinant virus is further infected into the insect cell, and the protein is added.
  • the gene transfer vector used in the method examples include pVL1392, pVL1393 (Betaton Dickinson) or pBlueBacIII (Invitrogen). And force S.
  • the baculovirus for example, the outrapha 'Californi power' nuclea, polyhedrosis virus, etc., which is a virus that infects the night stealing insects can be used.
  • the calcium phosphate method JP-A-2-227075
  • the lipofusion method [ Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)].
  • examples of expression vectors include Ti plasmids and tobacco mosaic virus vectors. Any promoter can be used as long as it can be expressed in plant cells. Examples thereof include the cauliflower mosaic virus (CaMV) 35S promoter and the actin 1 promoter.
  • CaMV cauliflower mosaic virus
  • As a method for introducing a recombinant vector into a plant cell any method can be used as long as it is a method for introducing DNA into a plant cell.
  • Agronocterium JP 59-140885; WO0080 / 00977
  • the Elect Mouth Position Method Japanese Patent Laid-Open No. 60-251887
  • a particle gun gene gun
  • DNA encoding a glycoprotein can be prepared as follows. Prepare total RNA or mRNA from various tissues or cells. CDNA is prepared from this total RNA or mRNA. Based on the amino acid sequence of the target glycoprotein, a degenerative primer is prepared, and a gene fragment encoding the target glycoprotein is obtained by PCR using the prepared cDNA as a saddle shape. In addition, using this gene fragment as a probe, a cDNA library can be screened to obtain DNA encoding the desired glycoprotein.
  • the human or non-human animal tissue or cell mRNA may be commercially available (eg, Clontech) or may be prepared from human or non-human animal tissue or cells as follows. Les.
  • Methods for preparing total RNA from human or non-human animal tissues or cells include guanidinium thiocyanate triacetoacetate method [Methods in Enzymology, 154, 3 (1987)] or acid thiocyanate / dicine-phenenole-chlorophenol. (AGPC) method [Analytical Biochemistry, 162 , 156 (1987); experimental medicine, £, 1937 (1991)].
  • AGPC acid thiocyanate / dicine-phenenole-chlorophenol.
  • Examples of a method for preparing mRNA from total R as poly (A) + RNA include oligo (dT) -immobilized cellulose column method (Molecular Cloning 2nd Edition).
  • mRNA can also be prepared by using a commercially available kit such as Fast Track mRNA Isolation Kit (Invitrogen) or Quick Prep RNA Purification Kit (GE Healthcare Bioscience).
  • a cDNA library is prepared from the prepared human or non-human animal tissue or cell mRNA.
  • One method for preparing a cDNA library is the method described in Molecular 'Cloning 2nd Edition, Current'Protocols' in 'Moleculars' Biology or A Laboratory Manual, 2nd Ed. (1989), or a commercially available kit. Examples thereof include a method using Superscript Plasmid System for cDNA Synthesis and Plasmid Cloning (Invitrogen) or ZAP-cDNA Synthesis Kit (Stratagene).
  • This cDNA library can be used as it is for the subsequent analysis, but the oligo cap method developed by Kanno et al. [Gene, 138. 171 (1994); Gene, 200. 149 (1997); Protein Nucleic Acid Enzyme, 1, 603 (1996); Experimental Medicine,, 2491 (1993); cDNA Cloning (Yodosha) (1996); Gene Library Construction The method (Yodosha) (1994)] may be used to prepare a cDNA library. Using a synthetic oligonucleotide or gene fragment obtained by PCR as a probe, colony hybridization from a cDNA library, certain hybrids, plaque hybridization (Molecular Cloning 2nd Edition), etc. Thus, cDNA of the target glycoprotein can be obtained.
  • DNA encoding the target glycoprotein can also be obtained by amplification using the PCR method.
  • the base sequence of the obtained DNA is a commonly used base sequence analysis method such as Sanger et al.'S dideoxy method [Proc. Nat 1. Acad. Sci. USA), 74, 5463 (1977)]! PRISM
  • Nucleotide sequence such as DNA Sequencer (Applied Biosystems) It can be determined by analyzing using an analyzer. Based on the determined DNA base sequence, a homology search program such as BLAST is used to search a base sequence database such as Genbank, EMBL, and DDBJ. It can also be confirmed that the gene encodes the target glycoprotein. Based on the determined DNA base sequence, the desired glycoprotein is encoded by chemical synthesis using a DNA synthesizer such as the DNA synthesizer model 392 (manufactured by Perkin'Elma Ichi) using the phosphoramidite method. You can also get the DNA you want.
  • a DNA synthesizer such as the DNA synthesizer model 392 (manufactured by Perkin'Elma Ichi) using the phosphoramidite method. You can also get the DNA you want.
  • the glycoprotein composition is produced and accumulated in yeast, animal cells, insect cells, or plant cells that can produce the glycoprotein composition by the above-described cell culture method.
  • yeast, animal cells, insect cells, or plant cells that can produce the glycoprotein composition by the above-described cell culture method.
  • Methods for producing glycoprotein compositions include intracellular production methods, extracellular secretion methods, and production methods on the outer membrane. The structure of the cells to be used and the glycoprotein molecules to be produced are determined. Select the method by changing the force S.
  • the target glycoprotein molecule can be actively secreted outside the host cell. Further, according to the method described in JP-A-2-227075, the production amount is increased by using a gene amplification system using a dihydrofolate reductase gene or the like.
  • a glycoprotein composition produced by a cell producing a glycoprotein composition is, for example, a glycoprotein.
  • the white matter composition is expressed in a state of being dissolved in the cells, after culturing, the cells are collected by centrifugation and suspended in an aqueous buffer, and then an ultrasonic crusher, French press, Mantongaurin homogenizer or It can be obtained from a cell extract obtained by disrupting cells with dynomill or the like.
  • 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,
  • a purified preparation of glycoprotein composition can be obtained by using a single method or a combination of methods such as electrophoresis. Specifically, for example, a method using immobilization to palliffin chromatography can be mentioned [Thr
  • the glycoprotein composition When the glycoprotein composition is expressed by forming an insoluble substance in the cell, the cell is recovered, disrupted, and centrifuged to obtain a glycoprotein from the insoluble substance obtained as a precipitate fraction. A composition can be obtained. The insoluble matter is solubilized with a protein denaturing agent, diluted or dialyzed to return the glycoprotein composition to a normal three-dimensional structure, and then purified by the same purification method as described above. A standard can be obtained.
  • the glycoprotein composition When the glycoprotein composition is secreted extracellularly, the glycoprotein composition can be recovered in the culture supernatant. That is, the culture supernatant is obtained by treating the culture by a method such as centrifugation as described above, and the glycoprotein is obtained from the culture supernatant by using the same isolation and purification method as described above. A purified preparation of the composition can be obtained.
  • the present invention also involves culturing cells capable of producing a glycoprotein composition in a medium to which at least one substance selected from sialic acid, a sialic acid polymer, and an oligosaccharide containing sialic acid is added.
  • the present invention relates to a method for improving the amount of sialic acid added to the glycoprotein composition produced from the cells.
  • the method for culturing cells capable of producing a glycoprotein composition can be performed as described above.
  • the amount of sialic acid added to the glycoprotein composition can be measured using techniques such as glycan quantification and glycan structure analysis using a two-dimensional glycan map method.
  • Sialic acid can be determined by acid hydrolysis with trifluoroacetic acid to liberate sialic acid and quantify it by the following method.
  • a specific method there is a method using a sugar composition analyzer BioLC manufactured by Dionetas. BioL HPAEC—PAD Oigh- er performance—exchange chromatography—pulsed amperometnc detection). Liq. Chromatogr., 6, 1577 (1983)].
  • Sialic acid can also be quantified by fluorescence labeling with 1,2-diamino-1,4,5-methylenedioxybenzene (DMB). Specifically, a sample hydrolyzed according to a known method [Ana 1. Biochem., 164, 138 (1987)] is fluorescently labeled with DMB and quantified by HPLC analysis.
  • DMB 1,2-diamino-1,4,5-methylenedioxybenzene
  • the retention time or elution position of sugar chains by reverse phase chromatography is plotted on the X axis
  • the retention time or elution position of sugar chains by normal phase chromatography is plotted on the vertical axis. This is a method for estimating the sugar chain structure by comparing with the results of known sugar chains.
  • the glycoprotein composition is hydrazine-degraded to release the sugar chain from the glycoprotein molecule, and the fluorescent labeling of the sugar chain with 2-aminoviridine (hereinafter abbreviated as ⁇ ).
  • ⁇ 2-aminoviridine
  • Biochem. 197 (1984) followed by gel filtration to separate the sugar chain from excess PA reagent and perform reverse phase chromatography.
  • normal phase chromatography is performed on each peak of the separated sugar chain. Based on these results, plot on a 2D glycan map and compare with the glycan standard (manufactured by Takara Bio Inc.) [Anal. Biochem., 171, 73 (1988)].
  • the sugar chain structure can be estimated.
  • the culture method of the present invention it is possible to suppress the amount of N-glycolylneuraminic acid bound to a glycoprotein composition produced by a cell having the ability to produce a glycoprotein composition. . That is, the present invention cultivates cells having the ability to produce a glycoprotein composition in a medium to which at least one substance selected from sialic acid, a sialic acid polymer, and an oligosaccharide containing sialic acid is added. The present invention relates to a method for suppressing the amount of N-glycolylneuraminic acid bound to a glycoprotein composition produced from the cells.
  • the method for culturing cells capable of producing a glycoprotein composition can be performed as described above.
  • the amount of N-glycolylneuraminic acid bound to the glycoprotein composition was determined using the quantitative method and the two-dimensional sugar chain mapping method, as with the amount of sialic acid added to the glycoprotein composition. It can be measured using techniques such as sugar chain structure analysis.
  • the medium for expansion until the main culture is EX-CELL TM 302 medium (Sigma. Aldrich 'Fine Chemical (hereinafter referred to as SAFC)), Methotrexe HSAFC (hereinafter referred to as MTX).
  • SAFC Aldrich 'Fine Chemical
  • MTX Methotrexe HSAFC
  • a medium supplemented with 500 nmol / L and L-glutamine (manufactured by Wako Pure Chemical Industries, Ltd.) 0 ⁇ 875 g / L was used.
  • About 10-30% of the medium was placed in a 125 mL, 250 mL, or lOOOOmL volume of triangular flask (manufactured by Cojung), and the cell suspension was seeded at 3 ⁇ 10 5 cells / mL. Thereafter, the cells were cultured at 35 ° C. for 3 or 4 days, and subculture was performed several times until the number of cells necessary for seeding of the main culture was obtained.
  • the final concentration in the medium for expansion culture is 1 mmol / L, 5 mmol.
  • N-acetyl-neuraminic acid manufactured by Malkin Bio Inc.
  • a medium adjusted to around / kg, pH 7.1 was prepared in advance.
  • a medium without the above sialic acid was also prepared.
  • a 200 g / L aqueous solution of L glucose (manufactured by Wako Pure Chemical Industries, Ltd.) was added as a mode so that the L-glucose concentration in the culture solution was about 4 g / L.
  • the viable cell density and viability were 0.4% trypan blue solution (manufactured by Invitrogen), the dye exclusion method, and the protein production concentration was Method— Pair Antibody Set for ELISA of human Antithronbin.
  • ELISA was performed using antigen ATIII) (manufactured by Affinity Biologics).
  • the amount of N-acetylneuraminic acid and N-glycolylneuraminic acid added was 1,2-diamino-4,5-methylenedioxybenzene (DMB) for fluorescent labeling (TaKaRa sialic acid fluorescent labeling reagent kit, Takara Bio (Measured by HPLC).
  • DMB 1,2-diamino-4,5-methylenedioxybenzene
  • the following formula 2 is used to calculate the cumulative viable cell density
  • the following formula 3 is used to express the specific production rate (SPR) representing the protein amount rate produced by the unit cell per unit time.
  • SPR specific production rate
  • 4 was used to calculate the relative sialic acid addition rate (%)
  • the following formula 5 was used to calculate the relative N-glycolylneuraminic acid addition rate (%).
  • SPR g / (10 6 cells x day)] protein production concentration ( ⁇ g / mL) ⁇ cumulative viable cell density [(10 6 cells / 111 x day)
  • Relative sialic acid addition rate (%) Sialic acid addition amount in glycoprotein during sialic acid addition culture (number / molecule) ⁇ Sialic acid addition amount in glycoprotein during sialic acid addition-free culture (number / molecule) X 100
  • Figs. As shown in Fig. 1, the amount of sialic acid added to the sugar chain bound to the glycoprotein on the 14th day of culture increases depending on the concentration of N-acetylneuraminic acid added to the medium. -The relative sialic acid addition rate compared to 100% in the culture without acetylylneuraminic acid was over 190%. In addition, as shown in FIG. 2, even in cell culture using a medium supplemented with 100 mmol / L N-acetylneuraminic acid, which is the highest concentration, the viable cell density and viability up to the 14th day of the culture were increased.
  • the maximum viable cell density reached 4.4 ⁇ 10 6 cells / mL or more on the 8th day of culture.
  • the SPR indicated by the slope of the graph was not affected by the N-acetylneuraminic acid addition concentration, and the protein production concentration produced was The amount was about 160 to 200 mg / L, which was almost the same as that in the culture without addition of N-acetylneuraminic acid.
  • the following batch culture was performed in a 250 mL Erlenmeyer flask, and the effect of adding sialic acid dimer or colominic acid at the start of the culture was evaluated.
  • the final concentration of 0.5 mmol / L was added to the medium for expansion culture of Example 1 with a 5 mmol ZL cyanoleic acid tag [N-Acylyluraminic Acid, dimer (a, 2 ⁇ 8 ) (Manufactured by Nacalai Testa)], or a final concentration of 0.05 mmol / L or 0.5 mmol / L of colominic acid (Nacalai Testa) is added to bring the osmotic pressure to around 330 mOsm / kg, pH 7.1 A conditioned medium was prepared in advance.
  • sialic acids can be used as a medium and control with N-acetylneuraminic acid (manufactured by Kyowa Hakko Kogyo Co., Ltd.) with a final concentration of 5 mmol / L or 10 mmol / L.
  • An acid-free medium was also prepared. 50 mL of each medium was poured into a 250 mL Erlenmeyer flask (manufactured by Koyung). Cells prepared by expansion culture are seeded at 3.0 ⁇ 10 5 cells / mL in each of the main culture media, and then 35 ° C, 100 rpm, 5% CO.
  • a culture solution at the end of the culture (the 14th day of culture) is obtained, and the amount of sialic acid added to the end of the sugar chain and N-glycolylneuraminic acid added to the glycoprotein produced from the culture solution The amount was measured.
  • Fig. 4 shows the results when cultured with N-acetylneuraminic acid added
  • Fig. 5 shows the results when cultured with sialic acid dimer added
  • Fig. 6 shows the results when colominic acid is added. The results when cultured are shown. Even in cell culture using a medium supplemented with sialic acid dimer and colominic acid, the amount of sialic acid added to the sugar chain bound to the glycoprotein on the 14th day of culture was the same as when N-acetylethylneuraminic acid was added. It has been found that it has risen to the same or higher level. In either case, the relative cyanoleic acid addition rate was improved depending on the addition concentration.
  • ⁇ 2,3-Sialyl ratose (manufactured by Kyowa Hakko Kogyo Co., Ltd.) or ⁇ 2,6-Sialyl lathose (manufactured by Kyowa Hakko Kogyo Co., Ltd.) is used as the medium for expansion culture of Example 1.
  • Each medium was added so that the final concentration was 4 mmol / L or 20 mmol / L, and the osmotic pressure was adjusted to around 330 mO sm / kg and pH 7.1.
  • a medium supplemented with 4 mmol / L or 2 Ommol / L of N-acetylneuraminic acid manufactured by Kyowa Hakko Kogyo Co., Ltd.
  • the above sialic acids ⁇ 2,3-sialylatatos, ⁇ 2 , 6-sialyllactose or ⁇ -acetylneuraminic acid
  • 50 mL of each medium was poured into a 250 mL Erlenmeyer flask (Coujung).
  • L-dulose manufactured by Wako Pure Chemical Industries, Ltd.
  • aqueous solution was added so that the L-darcose concentration in the culture solution was about 4 g / L.
  • sialic acids on the 14th day of cell culture using 20 mmol / L ⁇ 2,3 sialino leratotase or medium supplemented with 20 mmol / L ⁇ 2,6- sialyl latatose The relative addition rate of glycolylneuraminic acid was 27% and 45%, respectively, compared with the non-additive culture as 100%.
  • sialidase activity on day 14 of culture was 0.04 U / mL in the control culture without sialic acid, but about 0.01 U / mL in the 20 mmol / L ⁇ 2,3 sialyl ratatoose-added medium. Was suppressed.
  • the medium for expansion culture of Example 1 As the medium for main culture, the medium for expansion culture of Example 1 was used. The osmotic pressure was adjusted to 330 mOsm / kg and pH 7.1, and about 5 OmL of medium was added to a 250 mL Erlenmeyer flask (manufactured by Coung). Then, the cell suspension was seeded at 3 ⁇ 10 5 cells / mL, and then cultured in a 35 ° C., 100 rpm, 5% CO environment for 14 days.
  • N-acetylmethylneuraminic acid manufactured by Kyowa Hakko Kogyo Co., Ltd.
  • culture without the above sialic acid was also performed.
  • sialic acid had to be added by the stationary phase (in this case, until the 11th day of culture). Regardless of the time of addition of sialic acid, no significant difference was observed in the viability and viability of cultured cells.
  • N-acetylneuraminic acid (manufactured by Kyowa Hakko Kogyo Co., Ltd.) is further added to the expansion culture medium of Example 1 at 20 mmol / L, 40 mmol / L, 60 mmol / L, 80 mmol / L or lOOmmol.
  • a medium prepared with osmotic pressure of 330 mOsm / kg and a pH of about 7.1 and a non-prepared medium were prepared, and about 50 mL of medium was added to a 250 mL Erlenmeyer flask (manufactured by Coning). Then, the cell suspension was seeded at 3 ⁇ 10 5 cells / mL, and then cultured in a 35 ° C., 100 rpm, 5% CO environment for 3 days.
  • the culture solution was collected on the third day, and the viable cell density (cells / mU was measured.
  • the growth rate (h— was calculated from the following formula 6 using the viable cell density on the third day of culture. (Formula 6)
  • N-acetylneuraminic acid manufactured by Kyowa Hakko Kogyo Co., Ltd.
  • a medium without the above-mentioned sialic acid was also prepared as a control.
  • 700 mL of each medium was poured into a 2000 mL bioreactor (Able).
  • Cells prepared by expansion culture were seeded at 3.0 10 5 cells / 111, and then cultured in an environment of 35 ° C., 85 rpm, pH 7.1 for 14 days.
  • amino acids (L-alanin 0 ⁇ 14 g / L, L-arginine monohydrochloride 0 ⁇ 47 g / L, L-asparagine monohydrate 0 ⁇ 16 g / L, L-argasine acid 0 ⁇ 17 g / L , L-cystine dihydrochloride 0 ⁇ 51g / L, L-glutamic acid 0 ⁇ 42g / L, L-gnoretamine 7.3g / L, glycine 0 ⁇ 17g / L, L-histidine monohydrochloride dihydrate 0 ⁇ 24g / L L, L—isoleucine 0 ⁇ 59 g / L, L—leucine 0.59 g / L, L—lysine monohydrochloride 0.82 g /: L, L—methionine 0.17 g /: L, L—phenenolealanine 0.
  • the culture broth was collected once a day, and the viable cell density (cells / mL), viability (%), and protein production concentration (mg / L) were measured.
  • the culture solutions on the 8th, 10th, 12th and 14th days were obtained, and the amount of sialic acid added at the end of the sugar chain bound to the glycoprotein produced in the culture solution and N-glyco The amount of rilneuramic acid addition was measured.
  • the relative sialic acid addition rate was over 160% when compared to 100% in the culture without N-acetylneuraminic acid.
  • the viable cell density up to the 14th day of culture and There was no significant decrease in survival rate.
  • the maximum viable cell density reached 1.0 ⁇ 10 7 cells / mL or more on the 10th day of culture.
  • a method for producing a glycoprotein composition comprising adding at least one substance selected from sialic acid, a sialic acid polymer and an oligosaccharide containing sialic acid to a medium, and a glycoprotein composition
  • a method for culturing cells to be produced, a method for improving the amount of sialic acid added to a glycoprotein composition produced from the cells, and an N-glycolylneune that binds to a glycoprotein composition produced from the cells A method for suppressing the amount of laminic acid added is provided.
  • the glycoprotein composition produced according to the present invention is useful as a pharmaceutical because the amount of sialic acid added is higher than that of conventional glycoprotein compositions and the N-glycolylneuraminic acid content is suppressed.

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Abstract

A process for producing a glycoprotein composition improved in the amount of sialic acid added to the sugar chain; a method of culturing a cell producing a glycoprotein composition improved in the amount of sialic acid added to the sugar chain; a method of improving the amount of sialic acid to be added to the sugar chain of a glycoprotein composition; and a method of reducing the content of N-glycolylneuraminic acid in a glycoprotein composition.

Description

明 細 書  Specification
糖蛋白質組成物の製造方法  Method for producing glycoprotein composition
技術分野  Technical field
[0001] 本発明は、シアル酸、シアル酸重合物およびシアル酸を含むオリゴ糖から選ばれる 少なくとも 1種類の物質を添加した培地中で細胞を培養することにより糖蛋白質組成 物を製造する方法、糖蛋白質組成物を生産する細胞の培養方法、該細胞より生産さ れる糖蛋白質組成物に結合するシアル酸の付加量を向上させる方法、および該細 胞より生産される糖蛋白質組成物に結合する N—グリコリルノィラミン酸の付加量を抑 制させる方法に関する。  [0001] The present invention relates to a method for producing a glycoprotein composition by culturing cells in a medium to which at least one substance selected from sialic acid, sialic acid polymer and oligosaccharide containing sialic acid is added, A method for culturing cells that produce a glycoprotein composition, a method for increasing the amount of sialic acid added to the glycoprotein composition produced from the cells, and a glycoprotein composition produced from the cells The present invention relates to a method for suppressing the addition amount of N-glycolylneuraminic acid.
背景技術  Background art
[0002] 近年、細胞を用いた有用物質の生産研究が広く行われている。とくに、動物細胞に よる物質生産の重要度は増大しており、チャイニーズハムスター卵巣組織由来細胞（ 以下、 CHO細胞と称す）、ノ、イブリドーマなどの動物細胞を宿主として得られた物質 は、微生物を用いた場合に比べて、生産された物質に結合している糖鎖構造がヒト 由来の糖鎖に近いという性質を有するため、糖鎖構造を有する物質の生産を行う宿 主細胞として動物細胞が用いられて!/、る (特許文献 1、特許文献 2)。  In recent years, research on production of useful substances using cells has been widely performed. In particular, the importance of substance production by animal cells is increasing. Substances obtained from animal cells such as Chinese hamster ovary tissue-derived cells (hereinafter referred to as CHO cells), wild cells, and hybridomas are microorganisms. Compared to the case where it is used, since the sugar chain structure bonded to the produced substance is close to that of human-derived sugar chains, animal cells are used as host cells for producing substances having a sugar chain structure. Used! /, Ru (Patent Document 1, Patent Document 2).
[0003] 糖鎖の異化および分化に関与する酵素の一つであるシァリダーゼは、ウィルス、細 菌、原虫などの微生物、脊椎動物などの各種組織に分布する。シァリダーゼは糖蛋 白質の糖鎖の非還元末端からシアル酸を除去する酵素であり、糖鎖分解の初発反 応を触媒するェキソ型 α—グリコシダーゼの一種である。近年、動物の内在性シァリ ダーゼが、糖鎖分解の最初の反応をつかさどることによって、糖鎖修飾されている生 体分子の異化分解を調節し、立体構造や受容体による認識機構、細胞接着や免疫 機構などに影響を与えるなどの、多くの重要な細胞機能を制御することが明ら力、とな つた (非特許文献 2、非特許文献 3)。  [0003] Sialidase, one of the enzymes involved in sugar chain catabolism and differentiation, is distributed in various tissues such as viruses, bacteria, protozoa, and other vertebrates. Sialidase is an enzyme that removes sialic acid from the non-reducing terminus of glycoprotein sugar chains, and is a type of exo-type α-glycosidase that catalyzes the initial reaction of glycolysis. In recent years, animal endogenous sialidase regulates catabolism of biomolecules that have been glycosylated by controlling the initial reaction of glycosylation. It has become clear that it controls many important cell functions, such as affecting the immune mechanism (Non-patent document 2, Non-patent document 3).
[0004] 物質生産における宿主のシァリダーゼ活性の制御については、哺乳類細胞株を宿 主に用いた生産において多数報告がある。医薬品として知られているエリスロボイエ チン、組織型プラスミノーゲンァクチベータ（t— PA)、インターフェロン類およびモノ クローナル抗体などの糖蛋白質は、主に CHO細胞やマウスミエローマ細胞などの宿 主細胞に糖蛋白質をコードする cDNAを安定的に組み込んで樹立した形質転換体 を培養することにより、細胞外に分泌生産され、培養液中に蓄積される。糖蛋白質の 製造方法は、糖蛋白質の培養液を得る工程、培養液から糖蛋白質を精製する工程 を含む。生産細胞から培養液中に分泌された糖蛋白質の糖鎖構造は、生産細胞が 有する糖鎖修飾機構により多様であるが、多くの場合、糖蛋白質の糖鎖構造の非還 元末端側は、負電荷を有するシアル酸残基によって修飾を受けることが知られている (非特許文献 2、非特許文献 3)。糖蛋白質を生産する細胞が有するシァリダーゼは、 生産細胞の細胞質より培養液中に漏出し、蓄積するため（非特許文献 4)、シァリダ ーゼが培養液中に蓄積する糖蛋白質に作用し、糖蛋白質に結合した糖鎖からシァ ル酸を脱離させる（非特許文献 5)。同様に、糖蛋白質の製造における培養工程にお いて、シァリダ一ゼが糖蛋白質に結合した糖鎖からシアル酸を脱離させることが明ら 力、となって!/、る（非特許文献 6)。 [0004] Regarding the control of host sialidase activity in substance production, there have been many reports on production using mammalian cell lines as the host. Erythroboietin, a tissue-type plasminogen activator (t-PA), interferons and mono Glycoproteins such as clonal antibodies are secreted and produced extracellularly by culturing transformants established by stably incorporating cDNA encoding the glycoprotein into host cells such as CHO cells and mouse myeloma cells. And accumulates in the culture medium. The method for producing glycoprotein includes a step of obtaining a culture solution of glycoprotein and a step of purifying the glycoprotein from the culture solution. The sugar chain structure of glycoprotein secreted from the production cell into the culture medium varies depending on the sugar chain modification mechanism of the production cell, but in many cases, the non-reducing terminal side of the sugar chain structure of the glycoprotein is It is known to be modified by a sialic acid residue having a negative charge (Non-patent document 2, Non-patent document 3). Since sialidase contained in cells producing glycoprotein leaks and accumulates in the culture medium from the cytoplasm of the production cells (Non-patent Document 4), it acts on the glycoprotein accumulated in the culture medium, resulting in sugar The sialic acid is eliminated from the sugar chain bound to the protein (Non-patent Document 5). Similarly, in the culture process in the production of glycoprotein, it has become clear that sialidase can desorb sialic acid from the sugar chain bound to the glycoprotein (Non-Patent Document 6). ).
[0005] 医薬品として有用な糖蛋白質の多くは、患者の静脈中または皮下に、注射または 点滴などによって投与される力 血中濃度の維持には糖蛋白質の糖鎖の非還元末 端に付加されたシアル酸が重要な役割を果たしていることが知られている（非特許文 献 7)。シアル酸を脱離させた糖蛋白質の糖鎖構造は、非還元末端側にガラ外ース 残基が露出した構造体となり、該構造体が肝臓などに局在するァシァ口糖蛋白質受 容体 (ガラタトース受容体）に捕捉、分解されるため、シアル酸の脱離により糖蛋白質 の血中半減期は大きく低下する（非特許文献 8)。したがって、糖蛋白質医薬品の製 造において、糖蛋白質の糖鎖の非還元末端にシアル酸が付加された糖蛋白質を得 るために、生産細胞から漏出されるシァリダーゼの活性を抑制することが、糖蛋白質 医薬品を開発する上で重要であると考えられてレ、る。  [0005] Many glycoproteins useful as pharmaceuticals are added to the non-reducing end of the glycoprotein sugar chain to maintain the blood concentration in the patient's vein or subcutaneously by injection or infusion. It is known that sialic acid plays an important role (Non-patent Document 7). The glycoprotein structure of the glycoprotein from which sialic acid has been removed becomes a structure in which the non-reducing end side of the non-reducing end is exposed, and the structure is localized in the liver, etc. Since it is captured and decomposed by the galactose receptor), the blood half-life of glycoprotein is greatly reduced by the elimination of sialic acid (Non-patent Document 8). Therefore, in the production of glycoprotein pharmaceuticals, in order to obtain a glycoprotein in which sialic acid is added to the non-reducing end of the glycoprotein sugar chain, it is necessary to suppress the activity of sialidase leaked from production cells. Protein is considered important in the development of pharmaceuticals.
[0006] これまでに、シァリダーゼの活性を抑制する方法として、シァリダーゼ阻害剤を培地 に添加する糖蛋白質の製造方法が知られて!/、る（特許文献 3)。  [0006] To date, as a method for suppressing the activity of sialidase, a method for producing a glycoprotein in which a sialidase inhibitor is added to a medium has been known (Patent Document 3).
シァリダーゼ阻害剤として、シアル酸の一種である N ァセチルノイラミン酸やその 誘導体などが知られている（非特許文献 9)。このうち、 N ァセチルノイラミン酸、 2, 3 デヒドロー 2 デォキシ N ァセチルノイラミン酸（ 5 ァセタミドー 2 , 6 アンヒ ドロー 3, 5 ジデォキシ D グリセロー D ガラクト ノン 2 ェノン酸、 Neu5A c2enあるいは 2, 3 Dともいう）が CHO細胞由来のシァリダーゼも阻害することが知 られている（特許文献 3、非特許文献 4)。 As sialidase inhibitors, N-acetylneuraminic acid, which is a kind of sialic acid, and derivatives thereof are known (Non-patent Document 9). Of these, N-acetylmethylneuraminic acid, 2,3 dehydro-2 deoxy N-acetylmethylneuraminic acid (5-acetamido-2,6 It is known that draw 3,5 dideoxy D glycero D galactonone dienoic acid, also referred to as Neu5A c2en or 2, 3 D) also inhibits CHO cell-derived sialidase (Patent Document 3, Non-Patent Document 4).
[0007] さらに、 2, 3 デヒドロー 2 デォキシ N ァセチルノイラミン酸あるいは塩化銅を 培地へ添加し CHO細胞を培養することにより、生産される糖蛋白質からのシアル酸 の脱離を抑制する方法が知られている（非特許文献 10、非特許文献 1 1、特許文献 4 )。 [0007] Further, there is a method for suppressing detachment of sialic acid from a produced glycoprotein by adding 2,3 dehydro-2deoxy N-acetylneuraminic acid or copper chloride to the medium and culturing CHO cells. It is known (Non-patent document 10, Non-patent document 11 and Patent document 4).
しかしながら、シァリダーゼ阻害剤によっては培地へ溶解すると培地が酸性となるこ と、シァリダーゼ阻害剤によっては増殖阻害を起こすこと、適切な添加時期でないと 効果が無いこと、浸透圧が上昇してしまうこと、またシァリダーゼ阻害剤は高価である ため、高濃度のシァリダーゼ阻害剤を培地に添加すると高コストとなるなどの実用上 の問題点がある。  However, some sialidase inhibitors become acidic when dissolved in the medium, some sialidase inhibitors cause growth inhibition, are not effective unless they are added properly, and osmotic pressure increases. Moreover, since sialidase inhibitors are expensive, there are practical problems such as high costs when adding high concentrations of sialidase inhibitors to the medium.
[0008] その他に、糖蛋白質の糖鎖に、糖転移酵素などを用いて新たな糖鎖を付加させ、 糖蛋白質の糖鎖の構造を変化させることも知られている。例えば、シァリルトランスフ エラーゼにより、糖蛋白質の糖鎖の末端側にシアル酸を付加する方法が知られてい る（非特許文献 12)。シァリルトランスフェラーゼとしては《2→3シァリルトランスフェラ ーゼ（特許文献 5)、 a 2→8シァリルトランスフェラーゼ（特許文献 6)などが知られて いる。  In addition, it is also known to add a new sugar chain to a glycoprotein sugar chain using a glycosyltransferase or the like to change the structure of the glycoprotein sugar chain. For example, a method is known in which sialic acid is added to the terminal side of a sugar chain of a glycoprotein by sialyl transferase (Non-patent Document 12). As the sialyl transferase, << 2 → 3 sialyltransferase (Patent Document 5), a 2 → 8 sialyltransferase (Patent Document 6) and the like are known.
[0009] さらに、細胞により生産される糖蛋白質の糖鎖の組成を制御するために、細胞培養 における培地中の糖組成や糖濃度を変更することにより、糖蛋白質の糖鎖の種類を 変更する方法 (特許文献 7)、培地中の糖の比消費速度を制御することにより糖鎖構 造を改変する方法（特許文献 8)、培養細胞のシアル酸の細胞膜低透過性を利用し、 N ァセチルマンノサミン添加培地で細胞を培養して糖蛋白質中のシアル酸含量を 上昇させる方法（非特許文献 13)、細胞培養の培地へダルコサミンまたは N ァセチ ルダルコサミンを添加することにより、糖蛋白質の糖鎖へのガラクトース残基の転移を 抑制する方法（特許文献 9)、細胞培養の培地へアルカン酸またはその塩を添加し、 浸透圧または温度を制御することにより、糖蛋白質の糖鎖のシアル酸含量を向上さ せる方法（特許文献 10)、シァリダーゼ遺伝子をノックアウトした細胞を用いて糖蛋白 質を生産する方法 (特許文献 11)、細胞培養の培地における CO濃度をモニタリング [0009] Furthermore, in order to control the sugar chain composition of the glycoprotein produced by the cells, the type of glycoprotein sugar chain is changed by changing the sugar composition or sugar concentration in the medium in cell culture. Method (Patent Document 7), a method of modifying the sugar chain structure by controlling the specific consumption rate of sugar in the medium (Patent Document 8), and the low permeability of cultured cells to sialic acid. A method of increasing the sialic acid content in glycoproteins by culturing cells in a cetylmannosamine-added medium (Non-patent Document 13), and adding darcosamine or N-acetyldarcosamine to the cell culture medium. A method for suppressing the transfer of galactose residues to the chain (Patent Document 9), adding alkanoic acid or its salt to the cell culture medium, and controlling the osmotic pressure or temperature to control the sialic acid of the glycoprotein sugar chain Including How to improve (Patent Document 10), glycoprotein using knockout cells were Shiaridaze gene Quality monitoring (Patent Document 11), monitoring CO concentration in cell culture media
2  2
して、 CO濃度レベルを調整することにより培地中の N グリコリルノィラミン酸量を制  By adjusting the CO concentration level, the amount of N-glycolylneuraminic acid in the medium is controlled.
2  2
御する方法（特許文献 12)などが知られて!/、る。  How to control (Patent Document 12) is known!
[0010] 哺乳動物においては、シアル酸として N グリコリルノィラミン酸あるいは N ァセチ ルノイラミン酸が主に存在する（非特許文献 14)。しかし、ヒト正常組織においては、 C MP ァセチルノイラミン酸から、 CMP グリコリルノィラミン酸への転換酵素を欠損 して!/、るために N グリコリノレノイラミン酸は存在しな!/、（非特許文献 15)。再生医療 分野においてはヒト ES細胞培養時に使用される動物由来のフィーダ一や血清による N グリコリルノィラミン酸の汚染問題が挙げられており、移植時の免疫応答が懸念さ れている（非特許文献 14)。  [0010] In mammals, N-glycolylneuraminic acid or N-acetylneuraminic acid is mainly present as sialic acid (Non-patent Document 14). However, in normal human tissues, N-glycolinorenolemic acid does not exist because of the lack of the conversion enzyme from CMP acetylenylamic acid to CMP glycolylneuraminic acid! / (Non-patent Document 15). In the field of regenerative medicine, there is a problem of contamination of N-glycolylneuraminic acid with animal-derived feeders and serum used in human ES cell culture, and there is concern about immune responses during transplantation (non-patented) Reference 14).
[0011] また、細胞培養による糖蛋白質医薬品の製造においては、糖蛋白質の糖鎖の非還 元末端にシアル酸として N グリコリルノィラミン酸が付加することが知られている（非 特許文献 16)。このため、ヒトでは N グリコリルノィラミン酸を含む糖鎖を、免疫系が 異質な糖鎖として認識し、抗体産生が起こる可能性が懸念されている。従って、ヒトに 糖蛋白質組成物を投与する場合は N グリコリルノィラミン酸が混在しない方が好ま しい。  [0011] In addition, in the production of glycoprotein pharmaceuticals by cell culture, it is known that N-glycolylneuraminic acid is added as a sialic acid to the non-reducing end of the glycoprotein sugar chain (Non-patent Document 16). ). For this reason, in humans, there is a concern that sugar chains containing N-glycolylneuraminic acid may be recognized as foreign sugar chains by the immune system and antibody production may occur. Therefore, when administering a glycoprotein composition to humans, it is preferable that N-glycolylneuraminic acid is not mixed.
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非特許文献 14: Nature Medicine, 11, 228 (2005)  Non-Patent Document 14: Nature Medicine, 11, 228 (2005)
非特許文献 15:Proc. Natl. Acad. Sci. , USA 100, 12045 (1998) 非特許文献 16:FEBS Lett. , 275, 9 (1990)  Non-Patent Document 15: Proc. Natl. Acad. Sci., USA 100, 12045 (1998) Non-Patent Document 16: FEBS Lett., 275, 9 (1990)
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
[0012] 本発明の目的は、糖鎖へのシアル酸付加量を向上させた糖蛋白質組成物の製造 方法を提供すること、糖鎖へのシアル酸付加量を向上させた糖蛋白質組成物を生産 する細胞の培養方法を提供すること、糖蛋白質組成物中の糖鎖へのシアル酸付加 量を向上させる方法を提供すること、および糖蛋白質組成物中の Ν—グリコリルノイラ ミン酸量を抑制させる方法を提供することにある。  [0012] An object of the present invention is to provide a method for producing a glycoprotein composition having an increased amount of sialic acid added to a sugar chain, and to provide a glycoprotein composition having an improved amount of sialic acid added to a sugar chain. To provide a method for culturing cells to be produced, to provide a method for improving the amount of sialic acid added to a sugar chain in a glycoprotein composition, and to suppress the amount of グ リ コ -glycolylneuraminic acid in a glycoprotein composition It is to provide a method.
課題を解決するための手段  Means for solving the problem
[0013] すなわち、本発明は、以下の（1)〜（32)に関する。  [0013] That is, the present invention relates to the following (1) to (32).
(1) 5〜200mmol/Lのシアル酸、 0. ；!〜 200mmol/Lのシアル酸重合物、；!〜 200mmol/Lのシアル酸を含むオリゴ糖から選ばれる少なくとも 1種類の物質を添 加した培地中で細胞を培養し、培養物中に糖蛋白質組成物を生成蓄積させ、該培 養物から該糖蛋白質組成物を採取することを特徴とする、糖蛋白質組成物の製造方 法。 (1) 5 to 200 mmol / L sialic acid, 0.;! To 200 mmol / L sialic acid polymer; and! To 200 mmol / L sialic acid-containing oligosaccharides are added. A method for producing a glycoprotein composition comprising culturing cells in an added medium, producing and accumulating the glycoprotein composition in the culture, and collecting the glycoprotein composition from the culture. .
(2) シアル酸が N—ァセチルノイラミン酸である、（1)に記載の方法。  (2) The method according to (1), wherein the sialic acid is N-acetylneuraminic acid.
(3) シアル酸、シアル酸重合物およびシアル酸を含むオリゴ糖から選ばれる少なく とも 1種類の物質を培養開始時、対数増殖期、もしくは静止期に培地へ添加する、 (1 )または（2)に記載の方法。  (3) At least one substance selected from sialic acid, sialic acid polymer and oligosaccharide containing sialic acid is added to the medium at the start of culture, logarithmic growth phase, or stationary phase, (1) or (2 ) Method.
(4) 細胞を浸透圧 250〜400mOsm/kgで培養する、（1)〜（3)のいずれか 1項 に記載の方法。  (4) The method according to any one of (1) to (3), wherein the cells are cultured at an osmotic pressure of 250 to 400 mOsm / kg.
(5) 細胞が動物細胞である、（1)〜（4)のいずれ力、 1項に記載の方法。  (5) The method according to any one of (1) to (4), wherein the cell is an animal cell.
(6) 動物細胞が、以下の（a)〜（1)からなる群から選ばれる細胞である、 (5)に記載 の方法。  (6) The method according to (5), wherein the animal cell is a cell selected from the group consisting of the following (a) to (1).
(a)チャイニーズノ、ムスター卵巣組織由来 CHO細胞；  (a) Chinese CHO, derived from Muster ovarian tissue;
(b)ラットミエ口一マ糸田月包ネ朱 YB2/3HL. P2. Gi l . 16Ag. 20糸田月包；  (b) Rat Mye Kouichi Ma Itohtsuki Parcel Zhu YB2 / 3HL. P2. Gi l. 16Ag.
(c)マウスミエローマ細胞株 NS0細胞；  (c) mouse myeloma cell line NS0 cells;
(d)マウスミエローマ細胞株 SP2/0— Agl4細胞；  (d) mouse myeloma cell line SP2 / 0—Agl4 cells;
(e)シリアンノ、ムスター腎臓組織由来 BHK細胞；  (e) Syrianno, Muster kidney tissue-derived BHK cells;
(f)抗体を産生するハイプリドーマ細胞；  (f) a hyperidoma cell producing the antibody;
(g)ヒト白血病細胞株ナマルバ細胞；  (g) human leukemia cell line Namalva cells;
(h)ヒト白血病細胞株 NM— F9細胞；  (h) human leukemia cell line NM-F9 cells;
(i)ヒト胚性網膜細胞株 PER. C6細胞；  (i) human embryonic retinal cell line PER. C6 cells;
(j)胚性幹細胞； (j) embryonic stem cells;
(k)受精卵細胞；  (k) a fertilized egg cell;
(1)ヒ卜 HEK293細胞。  (1) Herbal HEK293 cells.
(7) 細胞力 糖蛋白質をコードする DNAが導入された細胞である、（1)〜（6)のい ずれか 1項に記載の方法。  (7) Cell force The method according to any one of (1) to (6), wherein the cell is a cell into which a DNA encoding a glycoprotein has been introduced.
(8) 糖蛋白質がアンチトロンビンである、（1)〜（7)のいずれか 1項に記載の方法。  (8) The method according to any one of (1) to (7), wherein the glycoprotein is antithrombin.
(9) 5〜200mmol/Lのシアル酸、 0. ；!〜 200mmol/Lのシアル酸重合物、；!〜 200mmol/Lのシアル酸を含むオリゴ糖から選ばれる少なくとも 1種類の物質を添 加した培地中で培養することを特徴とする、糖蛋白質組成物を生産する細胞の培養 方法。 (9) 5 to 200 mmol / L sialic acid, 0.;! To 200 mmol / L sialic acid polymer; A method for culturing cells producing a glycoprotein composition, comprising culturing in a medium supplemented with at least one substance selected from oligosaccharides containing 200 mmol / L sialic acid.
(10) シアル酸が N—ァセチルノイラミン酸である、（9)に記載の方法。  (10) The method according to (9), wherein the sialic acid is N-acetylneuraminic acid.
(11) シアル酸、シアル酸重合物およびシアル酸を含むオリゴ糖から選ばれる少な くとも 1種類の物質を培養開始時、対数増殖期、もしくは静止期に培地へ添加する、 ( 9)または（10)に記載の方法。  (11) At least one substance selected from sialic acid, sialic acid polymer and oligosaccharide containing sialic acid is added to the medium at the start of culture, logarithmic growth phase, or stationary phase, (9) or ( The method according to 10).
(12) 細胞を浸透圧 250〜400mOsm/kgで培養する、（9)〜（； 11)のいずれか 1 項に記載の方法。  (12) The method according to any one of (9) to (; 11), wherein the cells are cultured at an osmotic pressure of 250 to 400 mOsm / kg.
(13) 細胞が動物細胞である、（9)〜（； 12)のいずれか 1項に記載の方法。  (13) The method according to any one of (9) to (; 12), wherein the cell is an animal cell.
(14) 動物細胞力 以下の（a)〜（l)からなる群から選ばれる細胞である、（13)に記 載の方法。  (14) Animal cell force The method according to (13), which is a cell selected from the group consisting of the following (a) to (l).
(a)チャイニーズノ、ムスター卵巣組織由来 CHO細胞；  (a) Chinese CHO, derived from Muster ovarian tissue;
(b)ラットミエ口一マ糸田月包ネ朱 YB2/3HL. P2. Gi l . 16Ag. 20糸田月包；  (b) Rat Mye Kouichi Ma Itohtsuki Parcel Zhu YB2 / 3HL. P2. Gi l. 16Ag.
(c)マウスミエローマ細胞株 NS0細胞；  (c) mouse myeloma cell line NS0 cells;
(d)マウスミエローマ細胞株 SP2/0— Agl4細胞；  (d) mouse myeloma cell line SP2 / 0—Agl4 cells;
(e)シリアンノ、ムスター腎臓組織由来 BHK細胞；  (e) Syrianno, Muster kidney tissue-derived BHK cells;
(f)抗体を産生するハイプリドーマ細胞；  (f) a hyperidoma cell producing the antibody;
(g)ヒト白血病細胞株ナマルバ細胞；  (g) human leukemia cell line Namalva cells;
(h)ヒト白血病細胞株 NM— F9細胞；  (h) human leukemia cell line NM-F9 cells;
(i)ヒト胚性網膜細胞株 PER. C6細胞；  (i) human embryonic retinal cell line PER. C6 cells;
(j)胚性幹細胞； (j) embryonic stem cells;
(k)受精卵細胞；  (k) a fertilized egg cell;
(1)ヒ卜 HEK293細胞。  (1) Herbal HEK293 cells.
(15) 細胞力 糖蛋白質をコードする DNAが導入された細胞である、（9)〜（； 14)の いずれか 1項に記載の方法。  (15) Cell force The method according to any one of (9) to (; 14), which is a cell into which DNA encoding a glycoprotein has been introduced.
(16) 糖蛋白質がアンチトロンビンである、（9)〜（； 15)のいずれか 1項に記載の方 法。 (17) 5〜200mmol/Lのシァノレ酸、 0. ；!〜 200mmol/Lのシァノレ酸重合物、 1 〜200mmol/Lのシアル酸を含むオリゴ糖から選ばれる少なくとも 1種類の物質を 添加した培地中で、糖蛋白質組成物を生産する能力を有する細胞を培養することを 特徴とする、該細胞より生産される糖蛋白質組成物に結合するシアル酸の付加量を 向上させる方法。 (16) The method according to any one of (9) to (; 15), wherein the glycoprotein is antithrombin. (17) Medium supplemented with at least one substance selected from 5 to 200 mmol / L cyanoleic acid, 0.;! To 200 mmol / L cyanoleic acid polymer, oligosaccharide containing 1 to 200 mmol / L sialic acid A method for improving the amount of sialic acid added to the glycoprotein composition produced from the cell, comprising culturing cells capable of producing the glycoprotein composition.
(18) シアル酸が N—ァセチルノイラミン酸である、（17)に記載の方法。  (18) The method according to (17), wherein the sialic acid is N-acetylneuraminic acid.
(19) シアル酸、シアル酸重合物およびシアル酸を含むオリゴ糖から選ばれる少な くとも 1種類の物質を培養開始時、対数増殖期、もしくは静止期に培地へ添加する、 ( 17)または（18)に記載の方法。  (19) At least one substance selected from sialic acid, sialic acid polymer and oligosaccharide containing sialic acid is added to the medium at the start of culture, logarithmic growth phase, or stationary phase, (17) or ( The method described in 18).
(20) 細胞を浸透圧 250〜400mOsm/kgで培養する、（17)〜（； 19)のいずれか (20) The cell is cultured at an osmotic pressure of 250 to 400 mOsm / kg, any of (17) to (; 19)
1項に記載の方法。 The method according to item 1.
(21) 細胞が動物細胞である、（17)〜（20)のいずれか 1項に記載の方法。  (21) The method according to any one of (17) to (20), wherein the cell is an animal cell.
(22) 動物細胞力 以下の（a)〜（l)からなる群から選ばれる細胞である、（21)に記 載の方法。  (22) Animal cell force The method according to (21), which is a cell selected from the group consisting of the following (a) to (l).
(a)チャイニーズノ、ムスター卵巣組織由来 CHO細胞；  (a) Chinese CHO, derived from Muster ovarian tissue;
(b)ラットミエ口一マ糸田月包ネ朱 YB2/3HL. P2. Gi l . 16Ag. 20糸田月包；  (b) Rat Mye Kouichi Ma Itohtsuki Parcel Zhu YB2 / 3HL. P2. Gi l. 16Ag.
(c)マウスミエローマ細胞株 NS0細胞；  (c) mouse myeloma cell line NS0 cells;
(d)マウスミエローマ細胞株 SP2/0— Agl4細胞；  (d) mouse myeloma cell line SP2 / 0—Agl4 cells;
(e)シリアンノ、ムスター腎臓組織由来 BHK細胞；  (e) Syrianno, Muster kidney tissue-derived BHK cells;
(f)抗体を産生するハイプリドーマ細胞；  (f) a hyperidoma cell producing the antibody;
(g)ヒト白血病細胞株ナマルバ細胞；  (g) human leukemia cell line Namalva cells;
(h)ヒト白血病細胞株 NM— F9細胞；  (h) human leukemia cell line NM-F9 cells;
(i)ヒト胚性網膜細胞株 PER. C6細胞；  (i) human embryonic retinal cell line PER. C6 cells;
(j)胚性幹細胞； (j) embryonic stem cells;
(k)受精卵細胞；  (k) a fertilized egg cell;
(1)ヒ卜 HEK293細胞。  (1) Herbal HEK293 cells.
(23) 細胞力 糖蛋白質をコードする DNAが導入された細胞である、（17)〜（22) のいずれか 1項に記載の方法。 (24) 糖蛋白質がアンチトロンビンである、（17)〜（23)のいずれ力、 1項に記載の方 法。 (23) Cell force The method according to any one of (17) to (22), wherein the cell is a cell into which DNA encoding a glycoprotein has been introduced. (24) The method according to item 1, wherein the glycoprotein is antithrombin (17) to (23).
(25) 5〜200mmol/Lのシ ノレ酸、 0. ；!〜 200mmol/Lのシ ノレ酸重合物、 1 〜200mmol/Lのシアル酸を含むオリゴ糖から選ばれる少なくとも 1種類の物質を 添加した培地中で、糖蛋白質組成物を生産する能力を有する細胞を培養することを 特徴とする、該細胞より生産される糖蛋白質組成物に結合する N—グリコリルノイラミ ン酸量を抑制させる方法。  (25) Add at least one substance selected from 5-200 mmol / L sinolic acid, 0.;!-200 mmol / L sinolic acid polymer, 1-200 mmol / L sialic acid-containing oligosaccharide A cell having the ability to produce a glycoprotein composition in a cultured medium, wherein the amount of N-glycolylneuraminic acid bound to the glycoprotein composition produced from the cell is suppressed. Method.
(26) シアル酸が N—ァセチルノイラミン酸である、（25)に記載の方法。  (26) The method according to (25), wherein the sialic acid is N-acetylneuraminic acid.
(27) シアル酸、シアル酸重合物およびシアル酸を含むオリゴ糖から選ばれる少な くとも 1種類の物質を培養開始時、対数増殖期、もしくは静止期に培地へ添加する、 ( 25)または（26)に記載の方法。  (27) At least one substance selected from sialic acid, sialic acid polymer and oligosaccharide containing sialic acid is added to the medium at the start of culture, logarithmic growth phase, or stationary phase, (25) or ( The method described in 26).
(28) 細胞を浸透圧 250〜400mOsm/kgで培養する、（25)〜（27)のいずれか (28) The cell is cultured at an osmotic pressure of 250 to 400 mOsm / kg, any of (25) to (27)
1項に記載の方法。 The method according to item 1.
(29) 細胞が動物細胞である、（25)〜（28)のいずれか 1項に記載の方法。  (29) The method according to any one of (25) to (28), wherein the cell is an animal cell.
(30) 動物細胞が、以下の（a)〜（1)からなる群から選ばれる細胞である、請求項（2 9)に記載の方法。  (30) The method according to (29), wherein the animal cell is a cell selected from the group consisting of the following (a) to (1).
(a)チャイニーズノ、ムスター卵巣組織由来 CHO細胞；  (a) Chinese CHO, derived from Muster ovarian tissue;
(b)ラットミエ口一マ糸田月包ネ朱 YB2/3HL. P2. Gi l . 16Ag. 20糸田月包；  (b) Rat Mye Kouichi Ma Itohtsuki Parcel Zhu YB2 / 3HL. P2. Gi l. 16Ag.
(c)マウスミエローマ細胞株 NS0細胞；  (c) mouse myeloma cell line NS0 cells;
(d)マウスミエローマ細胞株 SP2/0— Agl4細胞；  (d) mouse myeloma cell line SP2 / 0—Agl4 cells;
(e)シリアンノ、ムスター腎臓組織由来 BHK細胞；  (e) Syrianno, Muster kidney tissue-derived BHK cells;
(f)抗体を産生するハイプリドーマ細胞；  (f) a hyperidoma cell producing the antibody;
(g)ヒト白血病細胞株ナマルバ細胞；  (g) human leukemia cell line Namalva cells;
(h)ヒト白血病細胞株 NM— F9細胞；  (h) human leukemia cell line NM-F9 cells;
(i)ヒト胚性網膜細胞株 PER. C6細胞；  (i) human embryonic retinal cell line PER. C6 cells;
(j)胚性幹細胞； (j) embryonic stem cells;
(k)受精卵細胞；  (k) a fertilized egg cell;
(1)ヒ卜 HEK293細胞。 (31) 細胞力 糖蛋白質をコードする DNAが導入された細胞である、（25)〜（30) のいずれか 1項に記載の方法。 (1) Herbal HEK293 cells. (31) Cell force The method according to any one of (25) to (30), wherein the cell is a cell into which DNA encoding a glycoprotein has been introduced.
(32) 糖蛋白質がアンチトロンビンである、（25)〜（31)のいずれ力、 1項に記載の方 法。  (32) The method according to any one of (25) to (31), wherein the glycoprotein is antithrombin.
発明の効果  The invention's effect
[0014] 本発明により、シアル酸、シアル酸重合物およびシアル酸を含むオリゴ糖から選ば れる少なくとも 1種類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 る糖蛋白質組成物の製造方法、および糖蛋白質生産細胞の培養方法、糖蛋白質組 成物中の糖鎖へのシアル酸付加量を向上させる方法、および糖蛋白質組成物中の N—グリコリルノィラミン酸量を抑制させる方法が提供される。本発明により製造される 糖蛋白質は、シアル酸、シアル酸重合物またはシアル酸を含むオリゴ糖を添加せず に培養して製造される糖蛋白質に対して、シアル酸付加量が増大し、 N—グリコリノレ ノィラミン酸含量が低減されており、医薬品として有用である。  [0014] According to the present invention, a glycoprotein composition characterized by culturing animal cells in a medium to which at least one substance selected from sialic acid, a sialic acid polymer and an oligosaccharide containing sialic acid is added. Production method, culture method for glycoprotein-producing cells, method for improving the amount of sialic acid added to the sugar chain in the glycoprotein composition, and suppression of the amount of N-glycolylneuraminic acid in the glycoprotein composition A method is provided. The glycoprotein produced according to the present invention increases the amount of sialic acid added to a glycoprotein produced by culturing without adding sialic acid, a sialic acid polymer or an oligosaccharide containing sialic acid. -Glycolinole neurolamic acid content is reduced and is useful as a medicine.
図面の簡単な説明  Brief Description of Drawings
[0015] [図 1]アンチトロンビン生産 CHO細胞株を用い、三角フラスコで、種々の濃度の N— ァセチルノイラミン酸を添加した培地を用いたフエドバツチ培養を行ったときの培養 1 4日目における相対シアル酸付加率を示す。  [0015] [Fig. 1] Cultivation of Fedbachii culture using antithrombin-producing CHO cell line and medium containing various concentrations of N-acetylneuraminic acid in Erlenmeyer flasks Day 4 The relative sialic acid addition rate in is shown.
[図 2]アンチトロンビン生産 CHO細胞株を用い、三角フラスコで、 N—ァセチルノイラ ミン酸を添加した培地を用いたフエドバツチ培養を行ったときの生細胞密度および生 存率を示す。生細胞密度は実線で、生存率は点線でそれぞれ示す。〇は N—ァセ チルノイラミン酸無添加対照群、 ·はN—ァセチルノイラミン酸 20mmol/L添加群、 △は N—ァセチルノイラミン酸 40mmol/L添加群、▲は N—ァセチルノイラミン酸 6 Ommol/L添加群、口は N—ァセチルノイラミン酸 80mmol/L添加群、國は N—ァ セチルノイラミン酸 100mmol/L添加群をそれぞれ示す。  FIG. 2 shows the viable cell density and viability when an antithrombin-producing CHO cell line was used and cultured in a Erlenmeyer flask and fed-batch culture using a medium supplemented with N-acetylneuraminic acid. Viable cell density is indicated by a solid line and viability is indicated by a dotted line. ◯ is a control group without addition of N-acetylneuraminic acid, · is a group with addition of 20 mmol / L of N-acetylneuraminic acid, △ is a group with addition of 40 mmol / L of N-acetylneuraminic acid, Laminic acid 6 Ommol / L added group, mouth shows N-acetylethylneuraminic acid 80 mmol / L added group, country shows N-acetylethylneuraminic acid 100 mmol / L added group.
[図 3]アンチトロンビン生産 CHO細胞株を用い、三角フラスコで N—ァセチルノイラミ ン酸を添加した培地を用いたフエドバツチ培養を行ったときの累積細胞密度対蛋白 質生産濃度を示す。〇は N—ァセチルノイラミン酸無添加対照群、 ·はN—ァセチ ルノイラミン酸 20mmol/L添加群、△はN—ァセチルノイラミン酸 40mmol/L添加 群、▲はN—ァセチルノイラミン酸 60mmol/L添加群、口は N—ァセチルノイラミン 酸 80mmol/L添加群、國は N—ァセチルノイラミン酸 100mmol/L添加群をそれ ぞれ示す。 [Fig. 3] Cumulative cell density versus protein production concentration when anti-thrombin-producing CHO cell line was used and fedbatch culture was performed in a Erlenmeyer flask supplemented with N-acetylneuraminic acid. 〇 is a control group without addition of N-acetylneuraminic acid, · is a group with addition of 20 mmol / L of N-acetylneuraminic acid, and △ is addition of 40 mmol / L of N-acetylneuraminic acid Group, ▲ is a group added with 60 mmol / L of N-acetylneuraminic acid, mouth is a group added with 80 mmol / L of N-acetylethylneuraminic acid, and country is a group added with 100 mmol / L of N-acetylethylneuraminic acid. Show.
[図 4]アンチトロンビン生産 CHO細胞株を用い、三角フラスコで N—ァセチルノイラミ ン酸を添加した培地を用いたフエドバツチ培養を行ったときの、培養 14日目における 相対シアル酸付加率を示す。  [Fig. 4] shows the relative sialic acid addition rate on the 14th day of culture when Fedbach culturing was performed using antithrombin-producing CHO cell line and medium supplemented with N-acetylneuraminic acid in an Erlenmeyer flask.
[図 5]アンチトロンビン生産 CHO細胞株を用い、三角フラスコでシアル酸ダイマー添 加を添加した培地を用いたフエドバツチ培養を行ったときの、培養 14日目における相 対シアル酸付加率を示す。  [Fig. 5] shows the relative sialic acid addition rate on the 14th day of culture when Fuedbatch culture was performed using antithrombin-producing CHO cell line and medium supplemented with sialic acid dimer in an Erlenmeyer flask.
[図 6]アンチトロンビン生産 CHO細胞株を用い、三角フラスコでコロミン酸を添加した 培地を用いたフエドバツチ培養を行ったときの、培養 14日目における相対シアル酸 付加率を示す。  FIG. 6 shows the relative sialic acid addition rate on the 14th day of culture when fed-batch culture was performed using antithrombin-producing CHO cell line and medium containing colominic acid in an Erlenmeyer flask.
[図 7]アンチトロンビン生産 CHO細胞株を用い、三角フラスコでシアル酸を含むオリゴ 糖を添加した培地を用いたフエドバツチ培養を行ったときの、培養 14日目における相 対シアル酸付加率を示す。口は N—ァセチルノイラミン酸添加群、譬は《2, 3—シァ リルラタトース添加群、盍は《2, 6—シァリルラタトース添加群をそれぞれ示す。  [Fig. 7] shows the relative sialic acid addition rate on the 14th day of culture when Fedbachii culture was performed using antithrombin-producing CHO cell line and medium containing oligosaccharide containing sialic acid in an Erlenmeyer flask. . The mouth indicates the N-acetylylneuraminic acid addition group, the 譬 indicates the << 2,3-sialyllatatose addition group, and the 盍 indicates the << 2,6-sialyllatatose addition group.
[図 8]アンチトロンビン生産 CHO細胞株を用い、三角フラスコでフエドバッチ培養を行 つたときの N—ァセチルノイラミン酸添加時期の違いによる相対シアル酸付加率を示 す。 [Fig. 8] shows the relative sialic acid addition rate according to the timing of N-acetylneuraminic acid addition when fed batch culture was performed in an Erlenmeyer flask using an antithrombin-producing CHO cell line.
[図 9]アンチトロンビン生産 CHO細胞株を用い、三角フラスコでフエドバッチ培養を行 つたときの N—ァセチルノイラミン酸添加時期の違いによる生細胞密度および生存率 を示す。〇は N—ァセチルノイラミン酸無添加対照群、 ·はN—ァセチルノイラミン酸 5日目添加群、△はN—ァセチルノイラミン酸 8日目添加群、▲はN—ァセチルノイラ ミン酸 9日目添加群、口は N—ァセチルノイラミン酸 10日目添加群、國は N—ァセチ ルノイラミン酸 11日目添加群、◊は N—ァセチルノイラミン酸 12日目添加群、 <^ N ーァセチルノイラミン酸 13日目添加群をそれぞれ示す。  [Fig. 9] shows the viable cell density and viability depending on the timing of N-acetylneuraminic acid addition when fed batch culture was performed in an Erlenmeyer flask using an antithrombin-producing CHO cell line. ◯ is a control group without addition of N-acetylneuraminic acid, · is a group with addition of N-acetylneuraminic acid on the 5th day, △ is a group with addition of N-acetylethylneuraminic acid on the 8th day, and ▲ is N-acetylethylneuraminine Acid 9-day addition group, mouth is N-acetylneuraminic acid 10-day addition group, country is N-acetylneuraminic acid 11-day addition group, ◊ is N-acetylneuraminic acid 12-day addition group, <^ N-acetylethyl neuraminic acid added on day 13 respectively.
園 10]アンチトロンビン生産 CHO細胞株を用い、三角フラスコで培地浸透圧を調整 した場合と調整していない場合の N—ァセチルノイラミン酸添加培養を行ったときの 増殖速度を示す。國は浸透圧調整群、口は浸透圧無調整群をそれぞれ示す。 10] Antithrombin production When CHO cell line was used, culture with N-acetylneuraminic acid added and not adjusted with medium osmotic pressure in Erlenmeyer flask The growth rate is shown. The country indicates the osmotic pressure adjustment group, and the mouth indicates the osmotic pressure non-adjustment group.
[図 11]アンチトロンビン生産 CHO細胞株を用い、バイオリアクターで、種々の濃度の N ァセチルノイラミン酸を添加した培地を用いたフエドバッチ培養を行ったときの、 N グリコリルノィラミン酸相対含量を示す。 N ァセチルノイラミン酸無添加培養 8日 目のシアル酸中に占める N グリコリルノィラミン酸含量を 100%として表示している 。 口は N ァセチルノイラミン酸無添加対照群、國は 20mmol/Lの N ァセチルノ イラミン酸添加群、△は 40mmol/Lの N ァセチルノイラミン酸添加群をそれぞれ 示す。 [Fig. 11] Relative content of N-glycolylneuraminic acid when fed-batch culture using antithrombin-producing CHO cell line and medium containing various concentrations of N-acetylneuraminic acid in a bioreactor Indicates. The content of N-glycolylneuraminic acid in the sialic acid on the 8th day after N-acetylethylneuraminic acid-free culture is shown as 100%. The mouth indicates the N-acetyl-neuraminic acid-free control group, the country indicates the 20-mmol / L N-acetyl-neuraminic acid-added group, and the Δ indicates the 40-mmol / L N-acetyl-neuraminic acid-added group.
[図 12]アンチトロンビン生産 CHO細胞株を用い、バイオリアクターで、種々の濃度の N ァセチルノイラミン酸を添加した培地を用いたフエドバッチ培養を行ったときの、 培養 14日目における相対シアル酸付加率を示す。斜線は N ァセチルノイラミン酸 無添加対照群、國は 20mmol/Lの N ァセチルノイラミン酸添加群、口は 40mmol /Lの N ァセチルノイラミン酸添加群をそれぞれ示す。  [FIG. 12] Relative sialic acid on day 14 of culture when fed batch culture using antithrombin-producing CHO cell line and using bioreactor with medium containing various concentrations of N-acetylneuraminic acid was performed. Indicates the rate of addition. The hatched lines indicate the N-acetyl-neuraminic acid-free control group, the country indicates the 20-mmol / L N-acetyl-neuraminic acid-added group, and the mouth indicates the 40-mmol / L-N-acetyl-neuraminic acid-added group.
[図 13]アンチトロンビン生産 CHO細胞株を用い、バイオリアクターで、種々の濃度の N ァセチルノイラミン酸を添加した培地を用いたフエドバッチ培養を行ったときの、 生細胞密度および生存率を示す。〇は N ァセチルノイラミン酸無添加対照群、國 は 20mmol/Lの N ァセチルノイラミン酸添加群、口は 40mmol/Lの N ァセチ ルノイラミン酸添加群をそれぞれ示す。  [FIG. 13] shows the viable cell density and viability when fed-batch culture was carried out in a bioreactor using antithrombin-producing CHO cell line and medium supplemented with various concentrations of N-acetylneuraminic acid. . 〇 indicates a control group without N-acetylneuraminic acid added, country indicates a group with 20 mmol / L N-acetylneuraminic acid added, and mouth indicates a group with 40 mmol / L N-acetylneuraminic acid added.
発明を実施するための最良の形態 BEST MODE FOR CARRYING OUT THE INVENTION
本発明は、シアル酸、シアル酸重合物およびシアル酸を含むオリゴ糖から選ばれる 少なくとも 1種類の物質を添加した培地中で細胞を培養し、培養物中に糖蛋白質組 成物を生成蓄積させ、該培養物から該糖蛋白質組成物を採取することを特徴とする 、糖蛋白質組成物の製造方法、および糖蛋白質生産細胞の培養方法に関する。 本発明において、シアル酸とは、 9つの炭素骨格からなるカルボキシル基を持つ 2 —ケト 3デォキシノン酸を意味し、ノィラミン酸ともいう。具体的には、 N ァセチノレ ノィラミン酸（Neu5Ac)、 N—グリコリルノィラミン酸（Neu5Gc)またはデァミノノイラミ ン酸 (KDN)、または、それらの誘導体などがあげられる。シアル酸誘導体としては、 上記のシアル酸がァセチル化、ラクチル化、硫酸化などの修飾を受けたものがあげら れ、具体的には、 N ァセチルー 4— O ノィラミン酸、 N ァセチルー 9— O ノイラ ミン酸、 N ァセチルー 8 , 9—ジ O ノイラミン酸、 N ァセチノレー 9 0—ラタトイ ノレノィラミン酸、 N ァセチル一 4— O ァセチル一 9—ラタトイルノイラミン酸、 N ァ セチルノイラミン酸一 9—リン酸、 N グリコロイル一 9— O ァセチルノイラミン酸、 N ダリコロイノレ 9 O ラタトイノレノィラミン酸、 N グリコロイノレノィラミン酸 8—石) ¾酸 、 5—アジドノイラミン酸、 N ァセチルー 9 ァセトアミドー 9ーデォキシノイラミン酸、 N ァセチルー 9 アジド 9 デォキシノイラミン酸または 5—アジドノイラミン酸な どがあげられる。 In the present invention, cells are cultured in a medium to which at least one substance selected from sialic acid, a sialic acid polymer and an oligosaccharide containing sialic acid is added, and a glycoprotein composition is produced and accumulated in the culture. In addition, the present invention relates to a method for producing a glycoprotein composition and a method for culturing a glycoprotein-producing cell, wherein the glycoprotein composition is collected from the culture. In the present invention, sialic acid means 2-keto-3-deoxynonic acid having a carboxyl group consisting of nine carbon skeletons, and is also called neuroamic acid. Specific examples include N-acetylenorelamic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc), diamineneuraminic acid (KDN), and derivatives thereof. Examples of sialic acid derivatives include those obtained by modifying the above sialic acid such as acetylation, lactylation, and sulfation. Specifically, N-acetylyl 4-O-neuraminic acid, N-acetylyl 9--O neuraminic acid, N-acetylyl 8,9-di-O neuraminic acid, N-acetylenole 9 0-ratato norenolamic acid, N-acetylyl 4-O Acetyl 9-ratatoylneuraminic acid, N-acetyl-neuraminic acid 9-phosphoric acid, N-glycolyl 9-O-acetylneuraminic acid, N Daricoleunor 9 O ratatoinorenolamic acid, N-glycolenorenolamic acid 8-stone) ¾ acid, 5-azidoneuraminic acid, N-acetyl-9-acetamido-9-deoxyneuraminic acid, N-acetyl-9-azido 9, deoxyneuraminic acid or 5-azidoneuraminic acid.
[0017] 本発明において、シアル酸重合物としては、上述の少なくとも 2分子のシアル酸また はシアル酸誘導体力 S、通常 α配置で 2→8または 2→9結合することにより得られたポ リマーであればいかなるものでもよぐ例えばコロミン酸、 Ν ァセチルノイラミン酸オリ ゴマーまたはそれらのナトリウム塩などがあげられる。具体的には、大腸菌 K1株から 誘導された 2→8 a結合単位を含み平均鎖長が 16単位であるコロミン酸または大腸 菌 K92株から誘導された 2→8結合シアル酸単位および 2→9結合シアル酸単位を 含み平均鎖長 78単位であるコロミン酸などがあげられる。 [0017] In the present invention, the sialic acid polymer is a polymer obtained by bonding 2 to 8 or 2 to 9 in the above-described at least two molecules of sialic acid or sialic acid derivative S, usually α configuration. Any one can be used, for example, colominic acid, acetylneuraminic acid oligomer, or a sodium salt thereof. Specifically, colominic acid having an average chain length of 16 units including 2 → 8 a binding units derived from E. coli K1 strain or 2 → 8 binding sialic acid units and 2 → 9 derived from Escherichia coli K92 strain Examples thereof include colominic acid having a combined chain sialic acid unit and an average chain length of 78 units.
[0018] 本発明において、シアル酸を含むオリゴ糖としては、例えばシアル酸の 5位のアミド 置換基がオリゴ糖で置換されたオリゴ糖結合型シアル酸類があげられる。具体的に は α 2, 3 シァリルラタトース、 α 2, 6 シァリルラタトース、ジシァリルラタトースまた はシァリルラタトサミンなどがあげられる。  In the present invention, examples of oligosaccharides containing sialic acid include oligosaccharide-linked sialic acids in which the amide substituent at the 5-position of sialic acid is substituted with an oligosaccharide. Specific examples include α 2,3 sialyl latatos, α 2,6 sialyl latatos, disialyl latatos or sialyl latatosamine.
本発明で用いられるシアル酸、シアル酸重合物またはシアル酸を含むオリゴ糖は、 化学合成による製造、遺伝子工学的手法等の公知の方法により得ることができる。ま た、市販のシアル酸、シアル酸重合物またはシアル酸を含むオリゴ糖を用いることも できる。市販のシアル酸、シアル酸重合物またはシアル酸を含むオリゴ糖の具体例と しては、コロミン酸ナトリウム塩、 Ν ァセチルノイラミン酸、 Ν グリコリルノィラミン酸、 Ν ァセチルノイラミン酸オリゴマーナトリウム塩または α 2, 6 シァリルラタトース（以 上、マルキンバイオ社製）などがあげられる。  The sialic acid, sialic acid polymer or oligosaccharide containing sialic acid used in the present invention can be obtained by a known method such as production by chemical synthesis or genetic engineering techniques. In addition, commercially available sialic acid, sialic acid polymer or oligosaccharide containing sialic acid can also be used. Specific examples of commercially available sialic acid, sialic acid polymer or oligosaccharide containing sialic acid include colominic acid sodium salt, ァ acetylneuraminic acid, Νglycolylneuraminic acid, Νacetylneuraminic acid oligomer Examples thereof include sodium salt and α 2,6 sialyl ratatose (hereinafter, Malkin Bio).
[0019] 本発明で用いられる、シアル酸等は、これまでに知られている培地に添加されるシ アル酸等に比べて高濃度で添加される。シアル酸の濃度は 5〜200mmol/L、好ま しくは 10〜； 150mmol/L、より好ましくは 20〜； 100mmol/Lである。シアル酸重合 物の濃度 (ま 0. ；!〜 200mmol/L、好ましく (ま 0. 2〜； 150mmol/L、より好ましく (ま 0 . 5〜； 100mmol/Lである。シアル酸を含むオリゴ糖の濃度は;!〜 200mmol/L、 好ましくは 2〜 150mmol/L、より好ましくは 5〜； 1 OOmmol/Lである。 [0019] The sialic acid or the like used in the present invention is added at a higher concentration than sialic acid or the like added to a medium known so far. The concentration of sialic acid is 5 ~ 200mmol / L, preferably 10 to 150 mmol / L, more preferably 20 to 100 mmol / L. Concentration of sialic acid polymer (from about 0.2 ;; to 200 mmol / L, preferably (from about 0.2 to 150 mmol / L, more preferably from about 0.5 to 100 mmol / L. Oligosaccharides containing sialic acid) The concentration of; is from !! to 200 mmol / L, preferably from 2 to 150 mmol / L, more preferably from 5 to 1 OOmmol / L.
[0020] 本発明において、シアル酸等を培地中に添加する時期としては、培養開始時、対 数増殖期、または静止期のいずれの期間でもよぐ複数の期間にまたがって添加し てもよい。対数増殖期とは、細胞培養において、細胞数が時間に対して対数的に増 加する時期をいう。静止期とは、対数増殖期の後、生細胞数の増加'維持から生存 率の低下をともなって減少に転じる時期以前の時期をいう。  [0020] In the present invention, sialic acid or the like may be added to the medium over a plurality of periods, such as at the start of culture, in the logarithmic growth phase, or in the stationary phase. . The logarithmic growth phase refers to a time when the number of cells increases logarithmically with time in cell culture. The quiescent phase is a period before the period when the number of living cells increases after the logarithmic growth phase and starts to decrease with a decrease in the survival rate.
[0021] 本発明において糖蛋白質組成物とは、非還元末端にシアル酸付加部位が存在す る糖鎖を有する糖蛋白質分子を含有する組成物であって、 N—グリコシド結合糖鎖ま たは O—グリコシド結合糖鎖を有する糖蛋白質分子からなる組成物をレ、う。糖蛋白質 分子は生産される細胞の糖鎖制御機構によって多様な糖鎖が結合するため、糖蛋 白質は、それらの多様な糖鎖構造を有する糖蛋白質分子の組成物となる。すなわち 、本発明において、糖蛋白質組成物とは、非還元末端にシアル酸付加部位が存在 する糖鎖を有し、 N—グリコシド結合糖鎖または O—グリコシド結合糖鎖を有する糖蛋 白質分子からなる組成物であれば、他の糖鎖構造はいかなるものであってもよい。  In the present invention, the glycoprotein composition is a composition containing a glycoprotein molecule having a sugar chain in which a sialic acid addition site is present at the non-reducing end, and is an N-glycoside-linked sugar chain or A composition comprising a glycoprotein molecule having an O-glycoside-linked sugar chain is used. Since glycoprotein molecules are linked to various sugar chains by the sugar chain control mechanism of the produced cells, the glycoprotein becomes a composition of glycoprotein molecules having these various sugar chain structures. That is, in the present invention, the glycoprotein composition is a glycoprotein molecule having a sugar chain having a sialic acid addition site at the non-reducing end and having an N-glycoside-bonded sugar chain or an O-glycoside-bonded sugar chain. Any other sugar chain structure may be used in the composition.
[0022] 本発明における細胞としては、糖蛋白質組成物を生産することができる細胞であれ ばいかなる細胞でもよぐ具体的には、酵母、昆虫細胞、植物細胞または動物細胞な どがあげられる力 S、好ましくは動物細胞があげられる。糖蛋白質組成物を生産するこ とができる細胞としては、糖蛋白質をコードする遺伝子を含有するベクターが導入さ れた形質転換細胞があげられる。糖蛋白質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は、例えば糖蛋白質をコードする DNAとプロモーターを 含む組換え体ベクターを、宿主となる上記細胞に導入することによって得ることがで きる。  [0022] The cells in the present invention may be any cells as long as they are capable of producing a glycoprotein composition, and specifically include the ability to include yeast, insect cells, plant cells, or animal cells. S, preferably animal cells. Examples of cells capable of producing a glycoprotein composition include transformed cells into which a vector containing a gene encoding a glycoprotein has been introduced. A transformed cell into which a vector containing a gene encoding a glycoprotein has been introduced can be obtained, for example, by introducing a recombinant vector containing a DNA encoding a glycoprotein and a promoter into the host cell. wear.
[0023] 酵母としては、サッカロミセス属、シゾサッカロミセス属、クリュイべ口ミセス属、トリコス ポロン属またはシュヮニォミセス属等に属する微生物、例えば、 Saccharomvces c erevisiae^ Schizosaccharomyces pomoe^ Kluyveromvc e s lactis. Tnchos poron pullulans, Schwanniomyces alluviusまたは Pichia nastorisなどをあ げること力 Sでさる。 [0023] Examples of the yeast include microorganisms belonging to the genus Saccharomyces, Schizosaccharomyces, Kluybe mouth mouth, Genus Trichospolon or Schneomyces, such as Saccharomvces c erevisiae ^ Schizosaccharomyces pomoe ^ Kluyveromvc es lactis. With the power S to raise poron pullulans, Schwanniomyces alluvius or Pichia nastoris.
[0024] 昆虫細胞としては、 Spodoptera frugiperdaの卵巣細胞である Sf 9、 Sf21 [カレ ント 'プロトコーノレズ 'イン'モレキュラー.ノ ィォロジ一； Baculovirus Expression Vectors, A Laboratory Manual, W. H. Freeman and company, New York (1992) Ίまたは Trichoplusianiの卵巣細胞である High 5 (インビト ロジェン社製）などをあげることができる。  [0024] Insect cells include Spodoptera frugiperda's ovarian cells Sf 9, Sf21 [Current 'Protocorenoles' in' Molecular. Neurology; Baculovirus Expression Vectors, A Laboratory Manual, WH Freeman and company, New York (1992) High 5 (manufactured by Invitrogen), which is an ovary cell of spider or Trichoplusiani, can be mentioned.
[0025] 植物細胞としては、タバコ、ジャガイモ、トマト、ニンジン、ダイズ、アブラナ、アルファ ノレファ、イネ、コムギ、ォォムギ、ヒメッリガネゴケまたはゥキクサなど由来の細胞をあ げること力 Sでさる。 [0025] As plant cells, it is possible to raise cells derived from tobacco, potatoes, tomatoes, carrots, soybeans, rapes, alfa norefas, rice, wheat, barley, green moss or duckweed with a force S.
動物細胞としては、ラット、マウス、ヒト、サル、ィヌまたはハムスターなど由来の細胞 、または、これらの動物細胞の雑種細胞系などがあげられる。動物細胞の具体的な 例としては、チャイニーズノヽムスター卵巣組織由来細胞株 CHO、 CHO-KKATC C CCL— 61)、 CHO/dhfr— (ATCC CRL— 9096)、 Pro5株（ATCC CRL — 1781)、 CHO— S (インビトロジェン社製 Cat # 11619)、マウス細胞株 NS0 (A TCC CRL— 1827)、 SP2/0 (ATCC CRL— 1581)、マウスミエローマ細胞株 S P2/0— Agl4、ラットミエローマ細胞株 Y3 Agl . 2. 3. (ATCC CRL— 1631)、 YO (ECACC Νο : 85110501) , YB2/3HL. Ρ2. Gi l . 16Ag. 20、 ΥΒ2/0 ( ATCC CRL— 1662)、シリアンハムスター腎臓組織由来細胞株 ΒΗΚ (ATCC C CL— 10)、 MDCK (ATCC CCL— 34)、ヒト細胞株 HEK293、 PER. C6™(EC ACC 96022940)、ヒト白血病細胞株ナマルバ細胞または NM— F9細胞あるいは 各動物種のハイプリドーマ細胞、胚性幹細胞または受精卵細胞などがあげられる。ま た、これらの株を無血清培地などに馴化させた亜株などであってもよぐさらにこれら の細胞に変異処理を施たり、ヒト以外の哺乳動物に抗原を免疫して取得された B細 胞と細胞融合することによって得られる細胞株であってもよい。  Examples of animal cells include cells derived from rats, mice, humans, monkeys, dogs, hamsters, etc., or hybrid cell lines of these animal cells. Specific examples of animal cells include Chinese nomstar ovarian tissue-derived cell lines CHO, CHO-KKATC C CCL—61), CHO / dhfr— (ATCC CRL—9096), Pro5 strain (ATCC CRL—1781), CHO — S (Invitrogen Cat # 11619), mouse cell line NS0 (A TCC CRL—1827), SP2 / 0 (ATCC CRL—1581), mouse myeloma cell line S P2 / 0— Agl4, rat myeloma cell line Y3 Agl 2. 3. (ATCC CRL— 1631), YO (ECACC Νο: 85110501), YB2 / 3HL. Ρ2. Gi l. 16Ag. 20, ΥΒ2 / 0 (ATCC CRL—1662), Syrian hamster kidney tissue-derived cell line AT (ATCC CCL—10), MDCK (ATCC CCL—34), human cell line HEK293, PER. C6 ™ (EC ACC 96022940), human leukemia cell line Namalva cells or NM-F9 cells, or hyperprideoma of each animal species Examples thereof include cells, embryonic stem cells, and fertilized egg cells. In addition, sub-strains obtained by acclimating these strains to a serum-free medium or the like may be used. These cells may be further mutated or immunized with non-human mammals. It may be a cell line obtained by cell fusion with a cell.
[0026] 本発明において、各種細胞を培養する方法としては、通常用いられる培養方法の いずれも用いることができる。例えば、バッチ培養、リピートバッチ培養、フエドバッチ 培養またはパーフュージョン培養などがあげられる力 フエドバツチ培養またはパーフ ユージョン培養を用いることが好ましい。フエドバツチ培養は、生理活性物質、栄養因 子等を連続的、または間欠的に少量ずつ追加供給する培養方法である。フエドバッ 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 所望の糖蛋白質組成物はバッチ培養で得られた場合に比べて高濃度であるため、 該糖蛋白質組成物の分離'精製が容易で、ノ ツチ培養に比べ、培地体積当たりの該 糖蛋白質組成物の生産量を増大させることができる。さらには、培地への添加溶液 中にシアル酸を加えておくことができるため、培養培地中のシアル酸濃度を制御する ことが容易である。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 率的に両者が分離され、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。本方法は、培養槽内の培養環境が常 に良好に保たれるため、好ましい。 In the present invention, as a method for culturing various cells, any of the commonly used culture methods can be used. For example, batch culture, repeat batch culture, fed-batch culture or perfusion culture. It is preferable to use Eugene culture. The Fuedbachi culture is a culture method in which physiologically active substances, nutrient factors, and the like are additionally supplied in small amounts continuously or intermittently. The fed-batch culture can prevent a decrease in the reached cell density of the cultured cells due to accumulation of waste products in the culture solution in which the metabolic efficiency of the cells is high. In addition, since the desired glycoprotein composition in the collected culture broth is at a higher concentration than that obtained in batch culture, the glycoprotein composition can be easily separated and purified, and compared to notch culture. In addition, the production amount of the glycoprotein composition per medium volume can be increased. Furthermore, since sialic acid can be added to the solution added to the medium, it is easy to control the sialic acid concentration in the culture medium. 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 fresh medium is newly supplied to the culture tank. Is the method. This method is preferable because the culture environment in the culture tank is always kept good.
[0027] 酵母の培養において用いられる培地としては、酵母が資化し得る炭素源、窒素源、 無機塩類などを含有し、形質転換体の培養を効率的に行える培地であれば、天然培 地、合成培地あるいは合成培地の機能を補う目的で天然物を添加した半合成培地 のいずれを用いてもよい。炭素源としては、酵母が資化し得るものであればよぐグノレ コース、フラクトースまたはスクロースなど、または、これらを含有する糖蜜、デンプン あるいはデンプン加水分解物などの炭水化物、酢酸またはプロピオン酸などの有機 酸、あるいは、エタノールまたはプロパノールなどのアルコール類などを用いることが できる。窒素源としては、アンモニア、塩化アンモニゥム、硫酸アンモニゥム、酢酸ァ ンモニゥムまたはリン酸アンモニゥムなどの無機酸もしくは有機酸のアンモニゥム塩、 その他の含窒素化合物、ペプトン、肉エキス、酵母エキス、コーンスチープリカー、力 ゼイン加水分解物、大豆粕または大豆粕加水分解物、あるいは、各種発酵菌体また はその消化物などを用いることができる。無機塩類としては、リン酸第一カリウム、リン 酸第二カリウム、リン酸マグネシウム、硫酸マグネシウム、塩化ナトリウム、硫酸第一鉄 、硫酸マンガン、硫酸銅または炭酸カルシウムなどを用いることができる。  [0027] As a medium used in yeast culture, any medium that contains a carbon source, nitrogen source, inorganic salts, and the like that can be assimilated by yeast, and that can efficiently culture transformants can be used. Either a synthetic medium or a semi-synthetic medium supplemented with natural products for the purpose of supplementing the function of the synthetic medium may be used. Examples of carbon sources include those that can be assimilated by yeast, such as glenolec, fructose or sucrose, or carbohydrates such as molasses, starch or starch hydrolysates, organic acids such as acetic acid or propionic acid. Alternatively, alcohols such as ethanol or propanol can be used. Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate or ammonium phosphate such as ammonium phosphate, other nitrogen-containing compounds, peptone, meat extract, yeast extract, corn steep liquor, strength Zein hydrolyzate, soybean meal or soybean meal hydrolyzate, various fermented bacterial cells, or digests thereof can be used. As the inorganic salt, monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, or calcium carbonate can be used.
[0028] 酵母の培養は、通常振盪培養または深部通気攪拌培養などの好気的条件下で行 う。培養温度は 15〜40°Cが好ましぐ培養時間は、通常 16時間〜 7日間である。培 養中の pHは通常 3· 0〜9· 0に保持する。 ρΗの調製は、無機または有機の酸、アル カリ溶液、尿素、炭酸カルシウム、アンモニアなどを用いて行う。また、培養中必要に 応じて、アンピシリンやテトラサイクリン等の抗生物質を培地に添加してもよい。また必 要に応じてインデューサーを培地に添加してもよい。 [0028] Yeast culture is usually performed under aerobic conditions such as shaking culture or deep aeration and agitation culture. The culture temperature is preferably 15 to 40 ° C, and the culture time is usually 16 hours to 7 days. Cultivation The pH during cultivation is usually maintained at 3 · 0 to 9 · 0. ρΗ is prepared using inorganic or organic acid, alkali solution, urea, calcium carbonate, ammonia, etc. Moreover, you may add antibiotics, such as an ampicillin and tetracycline, to a culture medium as needed during culture | cultivation. Moreover, you may add an inducer to a culture medium as needed.
[0029] 昆虫細胞の培養において用いられる培地としては、一般に使用されている ΤΝΜ— FH培地（ベタトン 'ディッキンソン社製）、 Sf— 900 II SFM培地（インビトロジェン 社製）、 EX— CELL™ 400、 EX— CELL™405 [いずれもシグマ'アルドリッチ'フ ァインケミカル（以下、 SAFCと称す）社製]または Grace ' s Insect Medium [Nat ure, 195, 788 (1962) ]などを用いることができる。  [0029] Common media used in insect cell culture are: ΤΝΜ—FH medium (Betaton 'Dickinson'), Sf—900 II SFM medium (Invitrogen), EX—CELL ™ 400, EX — CELL ™ 405 [both manufactured by Sigma's Aldrich 'Fine Chemical (hereinafter referred to as SAFC)] or Grace's Insect Medium [Natur, 195, 788 (1962)] can be used.
[0030] 昆虫細胞の培養は、通常 pH6〜7、 25〜30°C等の条件下で、；!〜 5日間行う。また 、培養中必要に応じて、ゲンタマイシン等の抗生物質を培地に添加してもよい。  [0030] Insect cells are usually cultured under conditions of pH 6 to 7, 25 to 30 ° C, etc .; for! To 5 days. Further, an antibiotic such as gentamicin may be added to the medium as needed during the culture.
植物細胞を培養する培地としては、一般に使用されているムラシゲ ·アンド 'スター グ（MS)培地またはホワイト（White)培地、あるいは、これら培地にオーキシン、サイ トカイニンなど、植物ホルモンを添加した培地などを用いることができる。  As a medium for culturing plant cells, commonly used Murashige & 'Sturg (MS) medium or White medium, or a medium in which plant hormones such as auxin and cytokinin are added to these mediums, etc. Can be used.
[0031] 植物細胞の培養は、通常 pH5〜9、 20〜40°Cの条件下で 3〜60日間行う。  [0031] Plant cells are usually cultured for 3 to 60 days under conditions of pH 5 to 9 and 20 to 40 ° C.
動物細胞の培養において用いられる培地としては、通常の動物細胞の培養に用い られる基礎培地であればいずれも用いることができる。具体的には、 RPMI1640培 ¾ L The Journal oi the American Medical Association, 199, 519 ( 1967)〕、 Eagleの MEM培地 [Science, 122. 501 (1952)〕、ダルベッコ改変 MEM培地 [Virology, 8 , 396 (1959)〕、 199培地 [Proceeding of the S ociety for the Biological Medicine, 73, 1 (1950)〕、 F12培地〔Proc. Any medium can be used as a medium used for culturing animal cells as long as it is a basal medium used for culturing ordinary animal cells. Specifically, RPMI1640 medium L The Journal oi the American Medical Association, 199, 519 (1967)], Eagle's MEM medium [Science, 122. 501 (1952)], Dulbecco's modified MEM medium [Virology, 8, 396 (1959)), 199 medium (Proceeding of the Society for the Biological Medicine, 73, 1 (1950)), F12 medium (Proc.
Natl. Acad. Sci. USA, 53, 288 (1965)〕、 IMDM培地 [J. Experim ental Medicine, 147, 923 (1978)〕、 DMEM培地、 Hybridoma Serum Free培地 (インビトロジェン社)、 Chemically Defined Hybridoma Serum F ree培地（インビトロジェン社）、 EX— CELL™— 302培地（SAFC社）または EX— C ELL™— CD— CHO (SAFC社)などが用いられる。また、必要に応じて動物細胞の 生育に必要な生理活性物質または栄養因子などを添加することができる。これらの 添加物は、培養前に予め培地に含有させる力、、さらに/または培養中に添加培地ま たは添加溶液として培養液へ適宜追加供給する。追加供給の方法は、 1溶液または 2種以上の混合溶液などいかなる形態でもよぐまた、添加方法は連続または断続の いずれでもよい。 Natl. Acad. Sci. USA, 53, 288 (1965)], IMDM medium [J. Experimental Medicine, 147, 923 (1978)], DMEM medium, Hybridoma Serum Free medium (Invitrogen), Chemically Defined Hybridoma Serum F Ree medium (Invitrogen), EX-CELL ™ -302 medium (SAFC) or EX-CELL ™ -CD-CHO (SAFC) is used. In addition, physiologically active substances or nutrient factors necessary for the growth of animal cells can be added as necessary. These additives can be added to the medium in advance before culturing and / or added to the medium during culturing. Alternatively, an additional solution is appropriately added to the culture medium. The additional supply method may be any form such as one solution or a mixed solution of two or more kinds, and the addition method may be continuous or intermittent.
[0032] 生理活性物質としては、インシュリン、 IGF—1、トランスフェリン、アルブミンまたは 補酵素 Q などがあげられる。栄養因子としては、糖、アミノ酸、ビタミン、加水分解物  [0032] Examples of the physiologically active substance include insulin, IGF-1, transferrin, albumin, and coenzyme Q. Nutritional factors include sugar, amino acids, vitamins and hydrolysates
10  Ten
または脂質などがあげられる。糖としては、グルコース、マンノースまたはフルクトース などがあげられ、 1種または 2種以上を組み合わせて用いられる。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—フエニノレアラニン、 L—プロリン、 Lーセリン、 L ースレオニン、 L—トリプトファン、 Lーチロシンまたは Lーバリンなどがあげられ、 1種 または 2種以上を組み合わせて用いられる。ビタミンとしては、 d ビォチン、 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、シァノコバラミンまたは DL— a トコフエロールなどがあげられ、 1 種または 2種以上を組み合わせて用いられる。加水分解物としては、大豆、小麦、米 、えんどう豆、とうもろこし、綿実、酵母抽出物などを加水分解したものがあげられる。 脂質としては、コレステロール、リノール酸またはリノレイン酸などがあげられる。また、 培養中必要に応じて、カナマイシン、ストレプトマイシン、ペニシリンまたはハイグロマ イシンなどの抗生物質を培地に添加してもよレ、。  Or a lipid etc. are mention | raise | lifted. Examples of the sugar include glucose, mannose, and fructose, and they are used alone or in combination of two or more. Amino acids include L-alanine, L-arginine, L-asparagin, L-aspartic acid, L-cystine, L--glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-fee Examples include ninorealanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine or L-valine, and are used alone or in combination of two or more. Vitamins include d biotin, D pantothenic acid, choline, folic acid, myo inositol, niacinamide, pyridoxal, riboflavin, thiamine, cyanocobalamin or DL-a tocopherol, and one or a combination of two or more. Used. Examples of the hydrolyzate include hydrolyzed soybeans, wheat, rice, peas, corn, cottonseed, yeast extract and the like. Examples of lipids include cholesterol, linoleic acid, and linolenic acid. In addition, antibiotics such as kanamycin, streptomycin, penicillin or hygromycin can be added to the medium as needed during culture.
[0033] 動物細胞を培養する際に、溶存酸素濃度制御、 pH制御、温度制御または攪拌な どは通常の各種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。 [0033] When cultivating animal cells, dissolved oxygen concentration control, pH control, temperature control, stirring, etc. can be performed according to the usual methods used for culturing various cells.
また、シアル酸等の酸性物質を培地に添加する場合には、 pHを細胞の成育およ び物質生産に適した中性域である pH5〜9、好ましくは pH6〜8に調整することが望 ましい。  In addition, when an acidic substance such as sialic acid is added to the medium, the pH should be adjusted to pH 5-9, preferably pH 6-8, which is a neutral range suitable for cell growth and substance production. Good.
[0034] 本発明において、細胞の培養や物質生産に適した浸透圧は 200〜600mOsm/ kgであり、好ましくは 250〜500mOsm/kgであり、より好ましくは 250〜400mOsm /kgである。  [0034] In the present invention, the osmotic pressure suitable for cell culture and substance production is 200 to 600 mOsm / kg, preferably 250 to 500 mOsm / kg, more preferably 250 to 400 mOsm / kg.
本発明にお!/、て、浸透圧 πは、 (式 1) In the present invention! /, The osmotic pressure π is (Formula 1)
π =RTiC  π = RTiC
(単位体積中の溶質のモル数： C、 R:気体定数、 T :絶対温度、 i :ファント ·ホッフの 係数)で表すことができる。  (The number of moles of solute in a unit volume: C, R: gas constant, T: absolute temperature, i: Fant-Hoff coefficient).
[0035] 浸透圧を測定する方法はいずれの方法でも良いが、凝固点降下法などがあげられ 本発明における培地中の浸透圧を制御する方法としては、培地中での浸透圧を調 整する物質（以下、浸透圧調整剤と称する）の濃度を変化させる方法、培地と異なる 浸透圧の溶液を培地へ添加する方法、浸透圧調整剤を溶解した溶液を培地へ添加 する方法、あるいは、培地中で生産される物質により培地のモル濃度または電解度 を変化させる方法などがあげられる。培地中での浸透圧調整剤の濃度を変化させる 方法としては、浸透圧調整剤を培地に添加する方法、または培地中の浸透圧調整剤 を除去する方法があげられる。浸透圧調整剤としては、培地の浸透圧に関与する物 質、すなわちモル濃度または電解度を変化させる物質であれば!/、かなるものでもよ いが、具体的には、塩化ナトリウム、塩化カリウムまたは塩化リチウムなどの塩類、グノレ コース、ガラクトース、マンノース、フコース、フノレクトース、シユークロース、マンニトー ル、キシロース、トレハロース、ソルビトールまたはグリセロールなどの糖類、各種アミノ 酸、ビタミン、大豆、小麦、米またはコーンなどの植物由来の加水分解物、ゥシ蛋白 質加水分解物などの動物由来蛋白質、イーストイクストラタトなどの酵母抽出物、水酸 化ナトリウム、炭酸ナトリウムまたは炭酸水素ナトリウムなど培地の pH調整に用いるァ ルカリ類、あるいは、補酵素 Q 、クレアチン、フイコール、酪酸などの培地添加物など Any method may be used for measuring the osmotic pressure, and examples include the freezing point depression method. The method for controlling the osmotic pressure in the medium in the present invention is a substance that adjusts the osmotic pressure in the medium. A method of changing the concentration of the osmotic pressure regulator (hereinafter referred to as an osmotic pressure regulator), a method of adding an osmotic pressure solution different from the culture medium, a method of adding a solution in which the osmotic pressure regulator is dissolved, or a medium containing And the method of changing the molar concentration or electrolysis of the medium depending on the substance produced in (1). Examples of the method for changing the concentration of the osmotic pressure adjusting agent in the medium include a method of adding the osmotic pressure adjusting agent to the medium and a method of removing the osmotic pressure adjusting agent in the medium. The osmotic pressure adjusting agent may be any substance that changes the osmotic pressure of the medium, that is, a substance that changes the molar concentration or the electrolysis degree. Specifically, sodium chloride, chloride may be used. Salts such as potassium or lithium chloride, sugars such as gnolecose, galactose, mannose, fucose, funolectose, sucrose, mannitol, xylose, trehalose, sorbitol or glycerol, various amino acids, vitamins, soybeans, wheat, rice or corn Plant-derived hydrolysates, animal-derived proteins such as ushi protein hydrolysates, yeast extracts such as yeast ixstrata, alkalis used to adjust the pH of media such as sodium hydroxide, sodium carbonate or sodium bicarbonate Or coenzyme Q, creatine, ficoll, butyric acid Medium additives such as
10  Ten
があげられるが、好ましくは塩化ナトリウムがあげられる。  Of these, sodium chloride is preferred.
[0036] 培地と異なる浸透圧の溶液を培地へ添加する方法としては、培地より浸透圧の高 V、溶液または浸透圧の低レ、溶液を添加する方法があげられる。浸透圧の高レ、溶液と しては、動物細胞に使用可能な培地に塩化ナトリウムなどの塩を 1種類または複数種 類添加した溶液、高濃度のアミノ酸またはビタミンなどにより構成された溶液、あるい は、動物細胞に使用可能な培地に塩、アミノ酸、ビタミンなどを加えた溶液などがあ げられる。浸透圧の低い溶液としては、動物細胞に使用可能な培地から塩化ナトリウ ム等の塩を 1種類または複数種類除去した溶液、アミノ酸、ビタミンなどの最小限の培 地成分により構成された溶液、動物細胞に使用可能な培地を水で希釈した溶液、あ るいは、水などがあげられる。 [0036] Examples of the method of adding a solution having an osmotic pressure different from that of the medium to the medium include a method of adding a solution having a higher osmotic pressure, a solution or a lower osmotic pressure, and a solution. Examples of high osmotic pressure solutions include solutions in which one or more salts such as sodium chloride are added to a medium that can be used for animal cells, and solutions that contain high concentrations of amino acids or vitamins. Or, a solution in which salts, amino acids, vitamins, etc. are added to a medium that can be used for animal cells. As a low osmotic pressure solution, sodium chloride is used from a medium usable for animal cells. A solution from which one or more salts such as amino acids are removed, a solution composed of a minimum of medium components such as amino acids and vitamins, a solution obtained by diluting a medium usable for animal cells with water, or water Etc.
[0037] 本発明の製造方法で得られる糖蛋白質組成物は、シアル酸等を培地に添加せず に細胞を培養して得られた糖蛋白質組成物と比較して、 1分子あたりのシアル酸付加 量が増加する。 1分子あたりのシアル酸付加量が増加した糖蛋白質組成物とは、糖 蛋白質分子のシアル酸付加部位の 50%以上、好ましくは 60%以上、より好ましくは 7 0%以上にシアル酸が結合された糖蛋白質組成物をいう。この糖蛋白質組成物は、 シアル酸等を培地に添加せずに得られた糖蛋白質組成物に比べて、生体内に投与 した場合の生理活性が向上する。生理活性としては、受容体との親和性、血中にお ける安定性、薬理活性または免疫原性などがあげられる。  [0037] The glycoprotein composition obtained by the production method of the present invention has a sialic acid per molecule as compared with a glycoprotein composition obtained by culturing cells without adding sialic acid or the like to the medium. The added amount increases. A glycoprotein composition having an increased amount of sialic acid per molecule means that sialic acid is bound to 50% or more, preferably 60% or more, more preferably 70% or more of the sialic acid addition site of the glycoprotein molecule. Glycoprotein composition. This glycoprotein composition has improved physiological activity when administered in vivo as compared to a glycoprotein composition obtained without adding sialic acid or the like to the medium. Examples of the physiological activity include affinity with a receptor, stability in blood, pharmacological activity or immunogenicity.
[0038] 本発明の方法で得られる糖蛋白質組成物は、糖鎖が結合した蛋白質組成物であ ればとくに限定はないが、好ましくは真核細胞由来糖蛋白質組成物などがあげられ、 より好ましくは哺乳動物細胞由来糖蛋白質組成物、さらに好ましくはヒト細胞由来糖 蛋白質組成物があげられる。具体的には以下の糖蛋白質からなる組成物があげられ る。抗体、エリスロポイエチン（EPO) . Biol. Chem. , 252. 5558 (1977 ) ]、トロンボポイエチン (TPO) [Nature, 369, 533 (1994) ]、組織型プラスミノ 一ゲンァクチベータ、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、 α 1ァ ンチトリプシン、 C1インヒビター、ハプトグロビン、活性化プロテイン C、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、血液凝固因子 XIII、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) . Biol. Chem. , 258, 9017 (1983) ]、マクロファー ジコロニー刺激因子（M— CSF) . Exp. Med. , 173, 269 (1992) ]、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. Bio 1. Chem. , 252, 1998 (1977) ]、インターフェロン α、インターフェロン /3、ィ ンターフェロン γ、インターロイキン一 2 (IL— 2) [Science, 193, 1007 (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、インターロイキン 12 (IL- 12) [J. 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 a、 Dnasel,ガラクトシダーゼ、 α—ダルコシダーゼ、グル コセレブロシダーゼ、ヘモグロビン、プロテイン S、トランスフェリンまたは静注用免疫 グロブリン (IVIG)などがあげられる。抗体としては、いかなる抗原結合性を有する抗 体でもよいが、腫瘍関連抗原に結合する抗体、アレルギーまた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、あるいは、ウィルスまたは細菌感染に関連する抗原 に結合する抗体であることが好ましぐ抗体のクラスは IgGが好ましい。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— 336 ( 1993)」、 JrLGD3irL体 [Cancer Immunol. Immunother. , 36 260— 2り ό, (1993) ]、抗 GM2抗体 [Cancer Res. , 54, 1511 - 1516, (1994) ]、抗 HER2抗体 [Proc. Natl. Acad. Sci. USA, 89, 4285— 4289 (1992 ) ]、抗 CD52抗体 [Nature, 332. 323— 327 (1988) ]、抗 MAGE抗体 [Briti sh J. Cancer, 83, 493— 497 (2000) ]、抗 HM1. 24抗体 [Molecular I mmunol. , 36, 387— 395 (1999) ]、抗副甲状腺ホルモン関連蛋白質（PTH rP)抗体 [Cancer, 88, 2909— 2911 , (2000) ]、抗 FGF8抗体 [Proc. Nat 1. Acad. Sci. USA, 86 , 9911 - 9915 (1989) ]、抗塩基十生繊糸隹芽糸田月包 増殖因子抗体、抗 FGF8受容体抗体 Biol. Chem. , 265. 16455— 164 63 (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 CI. Neurosci. Res. , 40, 647— 659 (1995) ]、抗インスリン様増 殖因子受容体抗体 Neurosci. Res. , 40, 647— 659 (1995) ]、抗 ΡΜ SA抗体 Urology, 160, 2396— 2401 (1998) ]、抗血管内皮細胞増殖 因子抗体 [Cancer Res. , 57, 4593— 4599 (1997) ]、抗血管内皮細胞増 殖因子受容体抗体 [Oncogene, 19, 2138— 2146 (2000) ]、抗 CA125抗体 、抗 17— 1A抗体、抗インテグリン 3抗体、抗 CD4抗体、抗 CD33抗体、抗 CD 22抗体、抗 HLA抗体、抗 HLA— DR抗体、抗 CD20抗体、抗 CD19抗体、抗 EGF 受容体抗体 [Immunology Today, 21 , 403— 410 (2000) ]、抗 HB— EGF 抗体、抗 Claudin4抗体、抗 Claudin3抗体、抗 PERP抗体、抗 SLC1A5抗体、抗 C CR4JTL体ま 7こ (ま i CDlOirL体 [American Journal of Clinical Pathology, 113. 374-382 (2000) ]などがあげられる。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としては、抗インターロイキン 6抗体 [Immunol. Rev. , 1 27, 5-24 (1992)]、抗インターロイキン 6受容体抗体 [Molecular Immunol. , 31, 371-381 (1994)]、抗インターロイキン 5抗体 [Immunol. Rev. , 1 27, 5-24 (1992)]、抗インターロイキン 5受容体抗体、抗インターロイキン 4抗 体 [Cytokine, 3, 562— 567 (1991) ]、抗インターロイキン 4受容体抗体 . Immunol. Meth. , 217, 41 50 (1998) ]、抗腫瘍壊死因子抗体 [Hybrid oma, 13, 183-190 (1994) ]、抗腫瘍壊死因子受容体抗体 [Molecular P harmacol. , 58, 237— 245 (2000) ]、抗 CCR4抗体 [Nature, 400. 77 6-780 (1999)]、抗ケモカイン抗体 . Immunol. Meth. , 174. 249— 257 (1994)]、抗ケモカイン受容体抗体 . Exp. Med. , 186, 1373— 13 81 (1997)]、抗 IgE抗体、抗 CD23抗体、抗 CDlla抗体 [Immunology Today , 21, 403-410 (2000)]、抗 CRTH2抗体 ϋ Immunol. , 162. 1278 -1286 (1999)]、抗 CCR8抗体（W099/25734)または抗 CCR3抗体（US62 07155)などがあげられる。循環器疾患に関連する抗原に結合する抗体としては、抗 GpIIb/lIIa抗体 CJ. Immunol. , 152. 2968 2976, (1994)]、抗血小板 由来増殖因子抗体 [Science, 253, 1129-1132, (1991) ]、抗血小板由来 増殖因子受容体抗体 ϋ· Biol. Chem. , 272, 17400— 17404, (1997)] または抗血液凝固因子抗体 [Circulation, 101, 1158-1164 (2000)]など 力 S挙げられる。乾癬、関節リウマチ、クローン病、潰瘍性大腸炎、全身性エリテマトー デス、多発性硬化症などの自己免疫疾患に関連する抗原に結合する抗体としては、 抗自己 DNA抗体 [Immunol. Letters, 72, 61— 68 (2000) ]抗 CD1 la抗 体、抗 ICAM3抗体、抗 CD80抗体、抗 CD2抗体、抗 CD3抗体、抗 CD4抗体、抗ィ ンテグリン α4 /37抗体、抗 CD40L抗体、抗 IL 2受容体抗体 [Immunology Tod ay, 21, 403-410 (2000) ]などが挙げられる。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としては、抗 gpl20抗体 [Structure, 8, 385— 3 95 (2000) ]、抗 CD4抗体 Rheumatology, 25, 2065— 2076, (199 8) ]、抗 CCR4抗体、抗ベロ毒素抗体 Clin. Microbiol. , 37, 396— 399 (1999) ]、などが挙げられる。 [0038] The glycoprotein composition obtained by the method of the present invention is not particularly limited as long as it is a protein composition to which a sugar chain is bound, and preferred examples include eukaryotic cell-derived glycoprotein compositions. Preferred is a mammalian cell-derived glycoprotein composition, and more preferred is a human cell-derived glycoprotein composition. Specific examples include compositions comprising the following glycoproteins. Antibody, erythropoietin (EPO). Biol. Chem., 252. 5558 (1977)], thrombopoietin (TPO) [Nature, 369, 533 (1994)], tissue-type plasminogen activator, prolokinase, thrombomodulin, anti Thrombin, α1-antitrypsin, C1 inhibitor, haptoglobin, activated protein C, blood coagulation factor VII, blood coagulation factor VIII, blood coagulation factor IX, blood coagulation factor X, blood coagulation factor XI, blood coagulation factor XII, blood Coagulation factor XIII, prothrombin complex, fibrinogen, albumin, gonadotropin, thyroid stimulating hormone, epidermal growth factor (EGF), hepatocyte growth factor (HGF), keratinocyte growth factor, activin, osteogenic factor, granulocyte colony sting Intense Factor (G-CSF). Biol. Chem., 258, 9017 (1983)], Macrophage Colony Stimulating Factor (M-CSF). Exp. Med., 173, 269 ( 1992)], stem cell factor (SCF), granulocyte-macrophage colony stimulating factor (GM-CSF) [J. Bio 1. Chem., 252, 1998 (1977)], interferon alpha, interferon / 3, interferon γ, interleukin 1 (IL-2) [Science, 193, 1007 (1976 ], Interleukin 6, Interleukin 10, Interleukin 11, Interleukin 12 (IL-12) [J. Leuc. Biol., 55, 280 (1994)], soluble interleukin 4 receptor, tumor necrosis factor a , Dnasel, galactosidase, α-darcosidase, glucocerebrosidase, hemoglobin, protein S, transferrin, or intravenous immunoglobulin (IVIG). The antibody may be any antigen-binding antibody, such as an antibody that binds to a tumor-related antigen, an antibody that binds to an antigen related to allergy or inflammation, an antibody that binds to an antigen related to cardiovascular disease, self IgG is the preferred class of antibody that preferably binds to an antigen associated with an immune disease or an antibody that binds to an antigen associated with a viral or bacterial infection. Anti-GD2 antibody [Anticancer Res., 13, 331-336 (1993)], JrLGD3irL body [Cancer Immunol. Immunother., 36 260-2 Ri., (1993)], GM2 antibody [Cancer Res., 54, 1511-1516, (1994)], anti-HER2 antibody [Proc. Natl. Acad. Sci. USA, 89, 4285-4289 (1992)], anti-CD52 antibody [Nature, 332. 323-327 (1988)], anti-MAGE antibody [Briti sh J. Cancer, 83, 493-497 (2000)], anti-HM1.24 antibody [Molecular Immunol., 36, 387-395 (1999)], anti Parathyroid hormone-related protein (PTH rP) antibody [Cancer, 88, 2909- 2911, (2000)], anti-FGF8 antibody [Proc. Nat 1. Acad. Sci. USA, 86, 9911-9915 (1989)], anti Basic Tensei-Fin Yaku Ita Tatsuki Growth Factor Antibody, Anti-FGF8 Receptor Antibody Biol. Chem., 265. 16455—164 63 (1990)], Anti-basic Fibroblast Growth Factor Receptor Antibody, Anti-Insulin-like Growth factor antibody CI. Neurosci. Res., 40, 647—659 (1995)], anti-insulin-like growth factor receptor antibody Ne urosci. Res., 40, 647—659 (1995)], anti-SA antibody Urology, 160, 2396—2401 (1998)], anti-vascular endothelial growth factor antibody [Cancer Res., 57, 4593—4599 (1997) )], Anti-vascular endothelial growth factor receptor antibody [Oncogene, 19, 2138— 2146 (2000)], anti-CA125 antibody, anti-17-1A antibody, anti-integrin 3 antibody, anti-CD4 antibody, anti-CD33 antibody, anti-antibody CD 22 antibody, anti-HLA antibody, anti-HLA—DR antibody, anti-CD20 antibody, anti-CD19 antibody, anti-EGF receptor antibody [Immunology Today, 21, 403-410 (2000)], anti-HB—EGF Antibodies, anti-Claudin4 antibodies, anti-Claudin3 antibodies, anti-PERP antibodies, anti-SLC1A5 antibodies, anti-CCR4JTL bodies (7 i CDlOirL bodies [American Journal of Clinical Pathology, 113. 374-382 (2000)], etc.) Anti-interleukin 6 antibody [Immunol. Rev., 127, 5-24 (1992)], anti-interleukin 6 receptor antibody [Molecular Immunol. 31, 371-381 (1994)], anti-interleukin 5 antibody [Immunol. Rev., 1 27, 5-24 (1992)], anti-interleukin 5 receptor antibody, anti-interleukin 4 antibody [Cytokine, 3 , 562-567 (1991)], anti-interleukin 4 receptor antibody. Immunol. Meth., 217, 41 50 (1998)], anti-tumor necrosis factor antibody [Hybrid oma, 13, 183-190 (1994)], Anti-tumor necrosis factor receptor antibody [Molecular P harmacol., 58, 237-245 (2000)], anti-CCR4 antibody [Nature, 400. 77 6-780 (1999)], anti-chemokine antibody. Immunol. Meth., 174 . 249-257 (1994)], anti-chemokine receptor antibody. Exp. Med., 186, 1373- 13 81 (1997)], anti-IgE antibody, anti-CD23 antibody, anti-CDlla antibody [Immunology Today, 21, 403-410 (2000)], anti-CRTH2 antibody ϋ Immunol., 162. 1278-1286 (1999)], anti-CCR8 antibody (W099 / 25734) or anti-CCR3 antibody (US62 07155), etc. Antigens related to cardiovascular diseases Anti-GpIIb / lIIa antibody CJ. Immunol., 152. 2968 2976, (1994)], antiplatelet-derived growth factor antibody [Science, 253, 1129-1132, (1991)], antiplatelet-derived antibodies Growth factor receptor antibody 抗体 · Biol. Chem., 272, 17400—17404, (1997)] or anticoagulant factor antibody [Circulation, 101, 1158-1164 (2000)]. Antibodies that bind to antigens associated with autoimmune diseases such as psoriasis, rheumatoid arthritis, Crohn's disease, ulcerative colitis, systemic lupus erythematosus, and multiple sclerosis include anti-self DNA antibodies [Immunol. Letters, 72, 61 — 68 (2000)] Anti-CD1 la antibody, anti-ICAM3 antibody, anti-CD80 antibody, anti-CD2 antibody, anti-CD3 antibody, anti-CD4 antibody, anti-integrin α4 / 37 antibody, anti-CD40L antibody, anti-IL 2 receptor antibody [Immunology Tod ay, 21, 403-410 (2000)]. Antibodies that bind to antigens associated with viral or bacterial infections include anti-gpl20 antibodies [Structure, 8, 385-3 95 (2000)], anti-CD4 antibody Rheumatology, 25, 2065-2076, (199 8)], anti-CCR4 antibody, anti-verotoxin antibody Clin. Microbiol., 37, 396-399 (1999)], etc. .
[0039] 本発明において、抗体とは、抗体の断片、 Fc領域を含む融合蛋白質を包含する。  [0039] In the present invention, an antibody includes an antibody fragment and a fusion protein containing an Fc region.
抗体としては、動物に抗原を免疫し、免疫動物の脾臓細胞より作製したハイブリド 一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 、抗体遺伝子を揷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。具体的には、ハイプリドーマが生産する抗体、ヒト型キメ ラ抗体、ヒト化抗体またはヒト抗体などをあげることができる。本発明において、ハイブ リドーマとは、ヒト以外の哺乳動物に抗原を免疫して取得された B細胞と、ラット、また はマウスに由来するミエローマ細胞とを細胞融合させて得られる細胞をいう。  Antibodies include antibodies produced by gene recombination techniques, in addition to antibodies secreted by hybridoma cells prepared from spleen cells of immunized animals, and antigen expression in which the antibody gene is inserted. Examples thereof include antibodies obtained by introducing a vector into a host cell. Specific examples include antibodies produced by Hypridoma, human chimeric antibodies, humanized antibodies or human antibodies. In the present invention, a hybridoma refers to a cell obtained by cell fusion of a B cell obtained by immunizing a mammal other than a human with an antigen and a myeloma cell derived from a rat or mouse.
[0040] 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ともいう）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ともレ、う）およびヒト抗体の軽鎖定常領域 (以下、 CLとも!/、う）とから なる抗体を意味する。ヒト以外の動物としては、マウス、ラット、ハムスターまたはラビッ トなど、ハイプ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。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揷入してヒト型キメラ抗体発現べクタ 一を構築し、宿主細胞へ導入することにより発現させ、製造すること力 Sできる。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 MgGl、 hi gG2、 MgG3、 MgG4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く、 κクラスあるいは λク ラスのものを用いることができる。 [0040] The human chimeric antibody is a non-human animal antibody heavy chain variable region (hereinafter, the heavy chain is referred to as H chain, the variable region is also referred to as HV or VH) and an antibody light chain variable region (hereinafter referred to as "H chain"). The light chain is also referred to as LV or VL as the 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! / )). As the non-human animal, any animal such as a mouse, rat, hamster or rabbit can be used as long as it can produce a hyperidoma. 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. Therefore, it is possible to construct a human chimeric antibody expression vector, introduce it into a host cell, express it and produce it. The CH of the human chimeric antibody may be any CH as long as it belongs to human immunoglobulin (hereinafter referred to as “hlg”), but the hlgG class is preferred, and MgGl, hi gG2, MgG3 belonging to the hlgG class are preferred. Any of the subclasses such as MgG4 can be used. The CL of the human chimeric antibody may be any as long as it belongs to hlg, and those of κ class or λ class can be used.
[0041」 ヒトイ匕抗体は、申目ネ甫十生決定領域、 complementarity determining region :以下 、 CDRという）移植抗体ともいう。ヒト化抗体は、ヒト以外の動物の抗体の VHおよび V Lの CDRのアミノ酸配列をヒト抗体の VHおよび VLの適切な位置に移植した抗体を 意味する。ヒト化抗体は、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 DNA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べ クタ一にそれぞれ揷入してヒト化抗体発現ベクターを構築し、該発現ベクターを宿主 細胞へ導入することにより発現させ、製造すること力 Sできる。ヒト化抗体の CHとしては 、 hlgに属すればいかなるものでもよいが、 MgGクラスのものが好適であり、さらに hlg Gクラスに属する MgGl、 MgG2、 MgG3または MgG4といったサブクラスのいずれ も用いること力 Sできる。また、ヒト化抗体の CLとしては、 hlgに属すればいかなるもので もよく、 κクラスまたはえクラスのものを用いることができる。 [0041] A human antibody is also referred to as a transplanted antibody (complementarity determining region: hereinafter referred to as CDR). Humanized antibodies are derived from VH and V of non-human animal antibodies. This refers to an antibody in which the amino acid sequence of the CDR of L is grafted to an appropriate position of human antibody VH and VL. Humanized antibodies are constructed by constructing DNA encoding the V region by grafting the VH and VL CDR sequences of non-human animal antibodies to the VH and VL CDR sequences of any human antibody. It is possible to construct a humanized antibody expression vector by introducing it into an expression vector for a host cell having a gene encoding CL, and to express it by introducing the expression vector into a host cell. . The CH of the humanized antibody may be any as long as it belongs to hlg, but the MgG class is preferable, and any of the subclasses such as MgGl, MgG2, MgG3, or MgG4 belonging to the hlg G class can be used. it can. Further, the CL of the humanized antibody may be any as long as it belongs to hlg, and those of κ class or e class can be used.
[0042] 本発明において、形質転換細胞を糖蛋白質組成物の生産に用いる場合、糖蛋白 質組成物を発現するベクターとしては、上記宿主細胞にぉレ、て自律複製可能なレ、し は染色体中への組込みが可能で、 目的とする糖蛋白質分子をコードする DNAを転 写できるプロモーターを含有して!/、るものが用いられる。酵母を宿主細胞として用い る場合、発現ベクターとしては、例えば、 YEP13 (ATCC 37115)、 YEp24 (ATC C 37051)または YCp50 (ATCC 37419)などをあげることができる。プロモータ 一としては、酵母菌株中で発現できるものであればいずれのものを用いてもよぐ例 えば、へキソースキナーゼ等の角军糖系の遺伝子のプロモーター、 PH05プロモータ 一、 PGKプロモーター、 GAPプロモーター、 ADHプロモーター、 gal 1プロモータ 一、 gal 10プロモーター、ヒートショック蛋白質プロモーター、 MF a 1 プロモーター または CUP 1プロモーターなどをあげることができる。酵母への組換えベクターの導 入方法としては、酵母に 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 [Methods, in Enzymol. , 194, 182 (19 90) ]、スフエロプラスト法 [Proc. Natl. Acad. Sci. U. S. A, 84, 1929 (1978) ]または酢酸リチウム法 . Bacteriology, 153, 163 (1983)； Pro c. Natl. Acad. Sci. U. S. A, 75, 1929 (1978) ]に記載の方法など をあげることができる。 [0042] In the present invention, when a transformed cell is used for production of a glycoprotein composition, the vector that expresses the glycoprotein composition may be a vector or a chromosome that can replicate autonomously in the host cell. Those that contain a promoter that can be incorporated into the DNA and that can transcribe DNA encoding the target glycoprotein molecule are used. When yeast is used as a host cell, examples of the expression vector include YEP13 (ATCC 37115), YEp24 (ATC C 37051), YCp50 (ATCC 37419), and the like. Any promoter can be used as long as it can be expressed in a yeast strain. Examples include promoters of cornose sugar genes such as hexose kinase, PH05 promoter 1, PGK promoter, GAP Promoter, ADH promoter, gal 1 promoter 1, gal 10 promoter, heat shock protein promoter, MF a 1 promoter or CUP 1 promoter. As a method for introducing a recombinant vector into yeast, any method can be used as long as it introduces DNA into yeast. For example, the Elect Mouth Position Method [Methods, in Enzymol., 194, 182 (19 90) ], Spheroplast method [Proc. Natl. Acad. Sci. US A, 84, 1929 (1978)] or lithium acetate method. Bacteriology, 153, 163 (1983); Pro c. Natl. Acad. Sci. , 75, 1929 (1978)].
[0043] 動物細胞を宿主として用いる場合、発現ベクターとして、例えば、 pcDNAI、 pcDM 8 (フナコシ社より市販）、 pAGE107 [特開平 3— 22979 ; Cytotechnology, 3, 133, (1990) ]、 pAS3— 3 (特開平 2— 227075)、 pCDM8 [Nature, 329, 840, (1987) ]、 pcDNAl/Amp (インビトロジェン社製）、 pREP4 (インビトロジ ェン社製）または PAGE103 . Biochemistry, 101 , 1307 (1987) ]などを あげること力 Sできる。プロモーターとしては、動物細胞中で発現できるものであればい 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 ea rly)遺伝子のプロモーター、 SV40の初期プロモーター、レトロウイルスのプロモータ 一、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または SR aプロモータ 一などをあげることができる。また、ヒト CMVの IE遺伝子のェンハンサーをプロモータ 一と共に用いてもよい。動物細胞への組換えベクターの導入方法としては、動物細 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、エレクトロポ レーシヨン法 [Cytotechnology, 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. U. S. A. , 84, 7413 (1987) ]、インジェクション法（マニピュレイティング.ザ.マウス.ェンブ リオ ·ァ ·ラボラトリ一 ·マニュアル）、パーティクルガン (遺伝子銃）を用いる方法（日本 特許第 2606856 ; 日本特許第 2517813)、 DEAE デキストラン法 [バイオマニュ アルシリーズ 4 遺伝子導入と発現'解析法（羊土社)横田崇 ·新井賢一編（1994) ] またはウィルスベクター法（マユピュレーティング'マウス'ェンブリオ第 2版）などをあ げること力 Sでさる。 [0043] When animal cells are used as hosts, expression vectors such as pcDNAI and pcDM 8 (commercially available from Funakoshi), pAGE107 [JP-A-3-22979; Cytotechnology, 3, 133, (1990)], pAS3-3 (JP-A-2-227075), pCDM8 [Nature, 329, 840, (1987)] , PcDNAl / Amp (manufactured by Invitrogen), pREP4 (manufactured by Invitrogen), or PAGE103. Biochemistry, 101, 1307 (1987)]. Any promoter can be used as long as it can be expressed in animal cells. For example, cytomegalovirus (CMV) IE (immediate ealy) gene promoter, SV40 early promoter, retrovirus promoter , Meta mouth thionein promoter, heat shock promoter or SRa promoter. An enhancer of the IE gene of human CMV may be used together with a promoter. As a method for introducing a recombinant vector into an animal cell, any method can be used as long as it introduces DNA into an animal cell. For example, the electroporation method [Cytotechnology, 3, 133 (1990)], calcium phosphate Method (Japanese Patent Laid-Open No. 2-227075), lipofusion method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)], injection method (manipulating the mouse mouse laboratory laboratory manual ), A method using a particle gun (Japanese Patent No. 2606856; Japanese Patent No. 2517813), DEAE Dextran Method [Biomanual Series 4 Gene Transfer and Expression Analysis Method (Yodosha) Takashi Yokota, Kenichi Arai (1994)] Or the virus vector method (Mayu Pulating 'Mouse' Embryo 2nd edition) etc.
昆虫細胞を宿主として用いる場合、例えばカレント'プロトコールズ 'イン ·モレキユラ 一 ·ノヽィォロシ一、 Baculovirus Expression Vectors, A Laboratory Manu al, W. H. Freeman and Company, New York (1992)または Bio/ Technology, 6, 47 (1988)などに記載された方法によって、蛋白質を発現す ること力 Sできる。即ち、組換え遺伝子導入ベクターおよび欠損型バキュロウィルスゲノ ムを昆虫細胞に共導入して昆虫細胞培養上清中に組換えウィルスを得た後、さらに 組換えウィルスを昆虫細胞に感染させ、蛋白質を発現させること力 Sできる。該方法に おいて用いられる遺伝子導入ベクターとしては、例えば、 pVL1392、 pVL1393 (ベ タトン 'ディッキンソン社製）または pBlueBacIII (インビトロジェン社製）などをあげるこ と力 Sできる。バキュロウィルスとしては、例えば、夜盗蛾科昆虫に感染するウィルスで あるアウトラファ 'カリフォルニ力'ヌクレア一 ·ポリへドロシス ·ウィルスなどを用いること ができる。組換えウィルスを調製するための、昆虫細胞への上記組換え遺伝子導入 ベクターと上記欠損型バキュロウィルスゲノムの共導入方法としては、例えば、リン酸 カルシウム法（特開平 2— 227075)またはリポフエクシヨン法 [Proc. Natl. Acad . Sci. U. S. A. , 84, 7413 (1987) ]などをあげることができる。 When insect cells are used as the host, for example, Current 'Protocols' in Molecula I. Noroiroshi, Baculovirus Expression Vectors, A Laboratory Manu al, WH Freeman and Company, New York (1992) or Bio / Technology, 6, 47 (1988) and the like, the ability to express proteins can be achieved. Specifically, a recombinant gene transfer vector and a defective baculovirus genome are co-introduced into an insect cell to obtain a recombinant virus in the insect cell culture supernatant, and then the recombinant virus is further infected into the insect cell, and the protein is added. Power to express S Examples of the gene transfer vector used in the method include pVL1392, pVL1393 (Betaton Dickinson) or pBlueBacIII (Invitrogen). And force S. As the baculovirus, for example, the outrapha 'Californi power' nuclea, polyhedrosis virus, etc., which is a virus that infects the night stealing insects can be used. As a method for co-introducing the above-described recombinant gene introduction vector and the above-described defective baculovirus genome into insect cells for preparing a recombinant virus, for example, the calcium phosphate method (JP-A-2-227075) or the lipofusion method [ Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)].
[0045] 植物細胞を宿主細胞として用いる場合、発現ベクターとして、例えば、 Tiプラスミド またはタバコモザイクウィルスベクターなどをあげることができる。プロモーターとして は、植物細胞中で発現できるものであればいずれのものを用いてもよぐ例えば、カリ フラワーモザイクウィルス（CaMV)の 35Sプロモーターまたはィネアクチン 1プロモー ターなどをあげることができる。植物細胞への組換えベクターの導入方法としては、 植物細胞に 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、ァグ ロノ クテリウム [特開昭 59— 140885 ; 特開昭 60— 70080 ; WO94/00977] , エレクト口ポレーシヨン法 [特開昭 60— 251887]またはパーティクルガン (遺伝子銃） を用いる方法 [日本特許第 2606856； 日本特許第 2517813]などをあげることが できる。 [0045] When plant cells are used as host cells, examples of expression vectors include Ti plasmids and tobacco mosaic virus vectors. Any promoter can be used as long as it can be expressed in plant cells. Examples thereof include the cauliflower mosaic virus (CaMV) 35S promoter and the actin 1 promoter. As a method for introducing a recombinant vector into a plant cell, any method can be used as long as it is a method for introducing DNA into a plant cell. For example, Agronocterium [JP 59-140885; WO0080 / 00977], the Elect Mouth Position Method [Japanese Patent Laid-Open No. 60-251887] or a method using a particle gun (gene gun) [Japanese Patent No. 2606856; Japanese Patent No. 2517813].
[0046] 本発明において、糖蛋白質をコードする DNAは、以下のように調製することができ る。各種組織または細胞から、全 RN Aまたは mRNAを調製する。この全 RNAまたは mRNAから cDNAを作製する。 目的とする糖蛋白質のアミノ酸配列に基づいて、デ ジエネレイティブプライマーを作製し、作製した cDNAを铸型として PCR法で目的と する糖蛋白質をコードする遺伝子断片を取得する。また、この遺伝子断片をプローブ として用い、 cDNAライブラリーをスクリーニングし、 目的とする糖蛋白質をコードする DNAを取得することもできる。ヒトまたは非ヒト動物の組織又は細胞の mRNAは巿販 のもの（例えばクローンテック社製）を用いてもよいし、以下のようにしてヒト又は非ヒト 動物の組織又は細胞から調製してもよレ、。ヒト又は非ヒト動物の組織又は細胞から全 RNAを調製する方法としては、チォシアン酸グァニジン トリフルォロ酢酸セシウム 法 [Methods in Enzymology, 154, 3 (1987) ]または酸性チォシアン酸グ /二シン-フエノーノレ-クロロホノレム (AGPC)法 [Analytical Biochemistry, 162 , 156 (1987)； 実験医学， £, 1937 (1991) ]などがあげられる。また、全 R ΝΑから poly(A) +RNAとして mRNAを調製する方法としては、オリゴ（dT)固定化 セルロースカラム法 (モレキュラー.クローニング第 2版）などがあげられる。さらに、 Fa st Track mRNA Isolation Kit (インビトロジェン社製）または Quick Prep m RNA Purification Kit (GEヘルスケア バイオサイエンス社製）などの市販のキッ トを用いることにより mRNAを調製することもできる。次に、調製したヒト又は非ヒト動 物の組織又は細胞 mRNAから cDNAライブラリーを作製する。 cDNAライブラリ一作 製法としては、モレキュラー 'クローニング第 2版、カレント'プロトコールズ'イン'モレ キユラ一'バイオロジーまたは A Laboratory Manual, 2 nd Ed. (1989)など に記載された方法、あるいは市販のキット、例えば Superscript Plasmid System for cDNA Synthesis and Plasmid Cloning (インビトロジェン社製）または Z AP-cDNA Synthesis Kit (ストラタジーン社製）を用いる方法などがあげられる 。この cDNAライブラリーをそのまま以降の解析に用いてもよいが、不完全長 cDNA の割合を下げ、なるべく完全長 cDNAを効率よく取得するために、菅野らが開発した オリゴキャップ法 [Gene, 138. 171 (1994)； Gene, 200. 149 (1997) ； 蛋白質核酸酵素， 1, 603 (1996)； 実験医学， , 2491 (1993)； cDNAクローニング（羊土社） （1996) ; 遺伝子ライブラリーの作製法（羊土社） （ 1994) ]を用いて cDNAライブラリーを調製してもよい。合成オリゴヌクレオチドや前 述の PCRで得られた遺伝子断片をプローブとして、 cDNAライブラリーからコロニー ハイブリダィゼーシヨン、あるレ、はプラークハイブリダィゼーシヨン（モレキュラー .クロ 一ユング第 2版）などにより、 目的とする糖蛋白質の cDNAを取得することができる。 また、 目的とする糖蛋白質をコードする遺伝子の 5'および 3'特異的プライマーを用 い、ヒト又は非ヒト動物の組織又は細胞に含まれる mRNAから合成した cDNAあるい は cDNAライブラリーを铸型として、 PCR法を用いて増幅することにより、 目的とする 糖蛋白質をコードする DNAを取得することもできる。取得した DNAの塩基配列は、 通常用いられる塩基配列解析方法、例えばサンガーらのジデォキシ法 [Proc. Nat 1. Acad. Sci. U. S. A. ) , 74, 5463 (1977) ]、ある!/、(ま ABI PRISM[0046] In the present invention, DNA encoding a glycoprotein can be prepared as follows. Prepare total RNA or mRNA from various tissues or cells. CDNA is prepared from this total RNA or mRNA. Based on the amino acid sequence of the target glycoprotein, a degenerative primer is prepared, and a gene fragment encoding the target glycoprotein is obtained by PCR using the prepared cDNA as a saddle shape. In addition, using this gene fragment as a probe, a cDNA library can be screened to obtain DNA encoding the desired glycoprotein. The human or non-human animal tissue or cell mRNA may be commercially available (eg, Clontech) or may be prepared from human or non-human animal tissue or cells as follows. Les. Methods for preparing total RNA from human or non-human animal tissues or cells include guanidinium thiocyanate triacetoacetate method [Methods in Enzymology, 154, 3 (1987)] or acid thiocyanate / dicine-phenenole-chlorophenol. (AGPC) method [Analytical Biochemistry, 162 , 156 (1987); experimental medicine, £, 1937 (1991)]. Examples of a method for preparing mRNA from total R as poly (A) + RNA include oligo (dT) -immobilized cellulose column method (Molecular Cloning 2nd Edition). Furthermore, mRNA can also be prepared by using a commercially available kit such as Fast Track mRNA Isolation Kit (Invitrogen) or Quick Prep RNA Purification Kit (GE Healthcare Bioscience). Next, a cDNA library is prepared from the prepared human or non-human animal tissue or cell mRNA. One method for preparing a cDNA library is the method described in Molecular 'Cloning 2nd Edition, Current'Protocols' in 'Moleculars' Biology or A Laboratory Manual, 2nd Ed. (1989), or a commercially available kit. Examples thereof include a method using Superscript Plasmid System for cDNA Synthesis and Plasmid Cloning (Invitrogen) or ZAP-cDNA Synthesis Kit (Stratagene). This cDNA library can be used as it is for the subsequent analysis, but the oligo cap method developed by Kanno et al. [Gene, 138. 171 (1994); Gene, 200. 149 (1997); Protein Nucleic Acid Enzyme, 1, 603 (1996); Experimental Medicine,, 2491 (1993); cDNA Cloning (Yodosha) (1996); Gene Library Construction The method (Yodosha) (1994)] may be used to prepare a cDNA library. Using a synthetic oligonucleotide or gene fragment obtained by PCR as a probe, colony hybridization from a cDNA library, certain hybrids, plaque hybridization (Molecular Cloning 2nd Edition), etc. Thus, cDNA of the target glycoprotein can be obtained. In addition, 5 'and 3' specific primers of the gene encoding the desired glycoprotein are used to synthesize cDNA or cDNA library synthesized from mRNA contained in tissues or cells of human or non-human animals. As described above, DNA encoding the target glycoprotein can also be obtained by amplification using the PCR method. The base sequence of the obtained DNA is a commonly used base sequence analysis method such as Sanger et al.'S dideoxy method [Proc. Nat 1. Acad. Sci. USA), 74, 5463 (1977)]! PRISM
377 DNAシークェンサ一（アプライド '·バイオシステムズ社製）などの塩基配列分 析装置を用いて分析することにより、決定すること力できる。決定した DNAの塩基配 列をもとに、 BLASTなどの相同性検索プログラムを用いて、 Genbank, EMBLおよ び DDBJなどの塩基配列データベースを検索することにより、取得した DNAがデー タベース中の遺伝子の中で目的とする糖蛋白質をコードしている遺伝子であることを 確認することもできる。決定された DNAの塩基配列に基づいて、フォスフォアミダイト 法を利用した DNA合成機 model 392 (パーキン 'エルマ一社製）などの DNA合成 機で化学合成することにより、 目的とする糖蛋白質をコードする DNAを取得すること もできる。 377 Nucleotide sequence such as DNA Sequencer (Applied Biosystems) It can be determined by analyzing using an analyzer. Based on the determined DNA base sequence, a homology search program such as BLAST is used to search a base sequence database such as Genbank, EMBL, and DDBJ. It can also be confirmed that the gene encodes the target glycoprotein. Based on the determined DNA base sequence, the desired glycoprotein is encoded by chemical synthesis using a DNA synthesizer such as the DNA synthesizer model 392 (manufactured by Perkin'Elma Ichi) using the phosphoramidite method. You can also get the DNA you want.
[0047] 本発明において上記の各種細胞の培養方法により、糖蛋白質組成物を生産するこ とが出来る酵母、動物細胞、昆虫細胞、あるいは植物細胞中に糖蛋白質組成物を生 成蓄積させ、該培養物より糖蛋白質組成物を採取することにより、糖蛋白質組成物を 製造すること力 Sできる。糖蛋白質組成物の生産方法としては、細胞内に生産させる方 法、細胞外に分泌させる方法、あるいは細胞外膜上に生産させる方法があり、使用 する細胞や、生産させる糖蛋白質分子の構造を変えることにより、該方法を選択する こと力 Sでさる。  [0047] In the present invention, the glycoprotein composition is produced and accumulated in yeast, animal cells, insect cells, or plant cells that can produce the glycoprotein composition by the above-described cell culture method. By collecting the glycoprotein composition from the culture, it is possible to produce the glycoprotein composition. Methods for producing glycoprotein compositions include intracellular production methods, extracellular secretion methods, and production methods on the outer membrane. The structure of the cells to be used and the glycoprotein molecules to be produced are determined. Select the method by changing the force S.
[0048] 糖蛋白質組成物が細胞内あるいは細胞外膜上に生産される場合、ポールソンらの 方法 . Biol. Chem. , 264. 17619 (1989) ]、ロウらの方法 [Proc. Nat 1. Acad. Sci. U. S. A. , 86, 8227 (1989)； Genes Develop. , 4 , 1288 (1990) ]、特開平 05— 336963または WO94/23021などに記載の方 法に準じて、糖蛋白質組成物を細胞外に積極的に分泌させることができる。すなわち 、細胞が形質転換細胞である場合には、遺伝子工学的手法を用いて発現ベクター に糖蛋白質分子をコードする DNAおよび糖蛋白質分子の発現に適切なシグナルぺ プチドをコードする DNAを揷入し、該発現ベクターを宿主細胞へ導入した後に糖蛋 白質分子を発現させることにより、 目的とする糖蛋白質分子を宿主細胞外に積極的 に分泌させること力 Sできる。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して生産量を上昇 させることあでさる。  [0048] When the glycoprotein composition is produced intracellularly or on the outer membrane, the method of Paulson et al. Biol. Chem., 264. 17619 (1989)], the method of Rowe et al [Proc. Nat 1. Acad Sci. USA, 86, 8227 (1989); Genes Develop., 4, 1288 (1990)], JP 05-336963 or WO94 / 23021, etc. Can be actively secreted. That is, when the cell is a transformed cell, DNA encoding a glycoprotein molecule and DNA encoding a signal peptide appropriate for expression of the glycoprotein molecule are inserted into an expression vector using genetic engineering techniques. By introducing the expression vector into the host cell and then expressing the glycoprotein molecule, the target glycoprotein molecule can be actively secreted outside the host cell. Further, according to the method described in JP-A-2-227075, the production amount is increased by using a gene amplification system using a dihydrofolate reductase gene or the like.
[0049] 糖蛋白質組成物を生産する細胞により製造された糖蛋白質組成物は、例えば糖蛋 白質組成物が細胞内に溶解した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またはダイノミルなどにより細胞を破砕することにより得られ た細胞抽出液から取得することができる。該細胞抽出液を遠心分離した後の上清か 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)—セファロースまたは DIAI ON HPA- 75 (三菱化学社製）などレジンを用いた陰イオン交換クロマトグラフィー 法、 S— Sepharose FF (GEヘルスケア バイオサイエンス社製）などのレジンを用 いた陽イオン交換クロマトグラフィー法、ブチルセファロースまたはフエ二ルセファロー 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、ァ フィニ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、あるいは等電点電気泳 動などの電気泳動法などの手法を単独あるいは組み合わせて用い、糖蛋白質組成 物の精製標品を得ることができる。具体的には、例えば、固定化へパリンァフィ二ティ 一クロマトグラフィーを用いた方法をあげることができる [Thromb. Res. , 5, 439 (1974) ; 続生化学実験講座 8、血液下巻（日本生化学会編）]。 [0049] A glycoprotein composition produced by a cell producing a glycoprotein composition is, for example, a glycoprotein. When the white matter composition is expressed in a state of being dissolved in the cells, after culturing, the cells are collected by centrifugation and suspended in an aqueous buffer, and then an ultrasonic crusher, French press, Mantongaurin homogenizer or It can be obtained from a cell extract obtained by disrupting cells with dynomill or the like. From the supernatant after centrifuging the cell 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 a resin such as ethyl (DEAE) —Sepharose or DIAI ON HPA-75 (Mitsubishi Chemical), and a positive ion using a resin such as S—Sepharose FF (GE Healthcare Bioscience) Ion exchange chromatography, hydrophobic chromatography using resins such as butyl sepharose or phenyl sepharose, gel filtration using molecular sieves, affinity chromatography, chromatofocusing, or isoelectric focusing A purified preparation of glycoprotein composition can be obtained by using a single method or a combination of methods such as electrophoresis. Specifically, for example, a method using immobilization to palliffin chromatography can be mentioned [Thromb. Res., 5, 439 (1974); Society))].
[0050] また、糖蛋白質組成物が細胞内に不溶体を形成して発現した場合は、細胞を回収 後破砕し、遠心分離を行うことにより、沈殿画分として得られた不溶体から糖蛋白質 組成物を得ることができる。該不溶体を蛋白質変性剤で可溶化し、希釈または透析 することにより、該糖蛋白質組成物を正常な立体構造に戻した後、上記と同様の単 離精製法により該糖蛋白質組成物の精製標品を得ることができる。  [0050] When the glycoprotein composition is expressed by forming an insoluble substance in the cell, the cell is recovered, disrupted, and centrifuged to obtain a glycoprotein from the insoluble substance obtained as a precipitate fraction. A composition can be obtained. The insoluble matter is solubilized with a protein denaturing agent, diluted or dialyzed to return the glycoprotein composition to a normal three-dimensional structure, and then purified by the same purification method as described above. A standard can be obtained.
[0051] 糖蛋白質組成物が細胞外に分泌された場合には、培養上清に該糖蛋白質組成物 を回収すること力できる。即ち、該培養物を上記と同様の遠心分離等の手法により処 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、糖蛋白質組成物の精製標品を得ることができる。  [0051] When the glycoprotein composition is secreted extracellularly, the glycoprotein composition can be recovered in the culture supernatant. That is, the culture supernatant is obtained by treating the culture by a method such as centrifugation as described above, and the glycoprotein is obtained from the culture supernatant by using the same isolation and purification method as described above. A purified preparation of the composition can be obtained.
また本発明は、シアル酸、シアル酸重合物、およびシアル酸を含むオリゴ糖から選 ばれる少なくとも 1種類の物質を添加した培地中で、糖蛋白質組成物を生産する能 力を有する細胞を培養することを特徴とする、該細胞より生産される糖蛋白質組成物 に結合するシアル酸の付加量を向上させる方法に関する。 [0052] 本発明のシアル酸の付加量を向上させる方法において、糖蛋白質組成物を生産 する能力を有する細胞を培養する方法は前述した通りに行うことができる。また、糖蛋 白質組成物へのシアル酸の付加量は、シアル酸定量および二次元糖鎖マップ法な どを用いた糖鎖構造解析などの手法を用いて測定することができる。シアル酸定量 は、トリフルォロ酢酸などで酸加水分解を行い、シアル酸を遊離し、以下の方法など により定量すること力 Sできる。具体的な方法としては、ダイオネタス社製糖組成分析装 置 BioLCを用いる方法があげられる。 BioL HPAEC— PAD O igh performan ce anion― exchange chromatography— pulsed amperometnc detection )法 . Liq. Chromatogr. , 6, 1577 (1983) ]に基づき糖鎖を分析する装 置である。また、 1 , 2—ジァミノ一 4, 5—メチレンジォキシ一ベンゼン（DMB)による 蛍光標識化法でもシアル酸を定量することができる。具体的には、公知の方法 [Ana 1. Biochem. , 164, 138 (1987) ]に従って酸加水分解した試料を DMBにより 蛍光ラベル化し、 HPLC分析することで定量することができる。 The present invention also involves culturing cells capable of producing a glycoprotein composition in a medium to which at least one substance selected from sialic acid, a sialic acid polymer, and an oligosaccharide containing sialic acid is added. The present invention relates to a method for improving the amount of sialic acid added to the glycoprotein composition produced from the cells. [0052] In the method for improving the amount of sialic acid added according to the present invention, the method for culturing cells capable of producing a glycoprotein composition can be performed as described above. In addition, the amount of sialic acid added to the glycoprotein composition can be measured using techniques such as glycan quantification and glycan structure analysis using a two-dimensional glycan map method. Sialic acid can be determined by acid hydrolysis with trifluoroacetic acid to liberate sialic acid and quantify it by the following method. As a specific method, there is a method using a sugar composition analyzer BioLC manufactured by Dionetas. BioL HPAEC—PAD Oigh- er performance—exchange chromatography—pulsed amperometnc detection). Liq. Chromatogr., 6, 1577 (1983)]. Sialic acid can also be quantified by fluorescence labeling with 1,2-diamino-1,4,5-methylenedioxybenzene (DMB). Specifically, a sample hydrolyzed according to a known method [Ana 1. Biochem., 164, 138 (1987)] is fluorescently labeled with DMB and quantified by HPLC analysis.
[0053] 糖蛋白質分子の糖鎖の構造解析は、 2次元糖鎖マップ法 [Anal. Biochem. ,  [0053] The structure analysis of sugar chains of glycoprotein molecules is based on the two-dimensional sugar map method [Anal. Biochem.
171 , 73 (1988)、生物化学実験法 23—糖蛋白質糖鎖研究法 (学会出版センタ 一）高橋禮子編 （1989) ]により行うことができる。 2次元糖鎖マップ法は、例えば、 X 軸には逆相クロマトグラフィーによる糖鎖の保持時間または溶出位置を、 Υ軸には順 相クロマトグラフィーによる糖鎖の保持時間または溶出位置をそれぞれプロットし、既 知糖鎖のそれらの結果と比較することにより、糖鎖構造を推定する方法である。具体 的には、糖蛋白質組成物をヒドラジン分解して、糖蛋白質分子から糖鎖を遊離し、 2 —アミノビリジン（以下、 ΡΑと略記する）による糖鎖の蛍光標識 . Biochem. , 9 5, 197 (1984) ]を行った後、ゲルろ過により糖鎖を過剰の PA化試薬などと分離 し、逆相クロマトグラフィーを行う。次いで、分取した糖鎖の各ピークについて順相クロ マトグラフィーを行う。これらの結果をもとに、 2次元糖鎖マップ上にプロットし、糖鎖ス タンダード（タカラバイオ社製） [Anal. Biochem. , 171 , 73 (1988) ]のスポ ットとの比較することにより、糖鎖構造を推定することができる。さらに各糖鎖の MAL DI— TOF— MSなどの質量分析を行い、 2次元糖鎖マップ法により推定される構造 を確言忍すること力 Sできる。 [0054] 本発明の培養方法を用いることにより、糖蛋白質組成物を生産する能力を有する 細胞により生産される糖蛋白質組成物に結合する N—グリコリルノィラミン酸量を抑制 させること力 Sできる。すなわち、本発明は、シアル酸、シアル酸重合物、およびシアル 酸を含むオリゴ糖から選ばれる少なくとも 1種類の物質を添加した培地中で、糖蛋白 質組成物を生産する能力を有する細胞を培養することを特徴とする、該細胞より生産 される糖蛋白質組成物に結合する N—グリコリルノィラミン酸量を抑制させる方法に 関する。 171, 73 (1988), Biochemical Experimental Method 23-Glycoprotein Glycan Research Method (Academic Publishing Center I), Takahashi Keiko (1989)]. In the two-dimensional glycan mapping method, for example, the retention time or elution position of sugar chains by reverse phase chromatography is plotted on the X axis, and the retention time or elution position of sugar chains by normal phase chromatography is plotted on the vertical axis. This is a method for estimating the sugar chain structure by comparing with the results of known sugar chains. Specifically, the glycoprotein composition is hydrazine-degraded to release the sugar chain from the glycoprotein molecule, and the fluorescent labeling of the sugar chain with 2-aminoviridine (hereinafter abbreviated as ΡΑ). Biochem. 197 (1984)], followed by gel filtration to separate the sugar chain from excess PA reagent and perform reverse phase chromatography. Next, normal phase chromatography is performed on each peak of the separated sugar chain. Based on these results, plot on a 2D glycan map and compare with the glycan standard (manufactured by Takara Bio Inc.) [Anal. Biochem., 171, 73 (1988)]. Thus, the sugar chain structure can be estimated. Furthermore, mass analysis such as MAL DI-TOF-MS of each glycan can be performed and the structure estimated by the two-dimensional glycan map method can be confirmed. [0054] By using the culture method of the present invention, it is possible to suppress the amount of N-glycolylneuraminic acid bound to a glycoprotein composition produced by a cell having the ability to produce a glycoprotein composition. . That is, the present invention cultivates cells having the ability to produce a glycoprotein composition in a medium to which at least one substance selected from sialic acid, a sialic acid polymer, and an oligosaccharide containing sialic acid is added. The present invention relates to a method for suppressing the amount of N-glycolylneuraminic acid bound to a glycoprotein composition produced from the cells.
[0055] 本発明の N—グリコリルノィラミン酸量の抑制させる方法において、糖蛋白質組成 物を生産する能力を有する細胞を培養する方法は前述した通りに行うことができる。 また、糖蛋白質組成物に結合する N—グリコリルノィラミン酸量は、前述の糖蛋白質 組成物へのシアル酸の付加量と同様、定量法および二次元糖鎖マップ法などを用 V、た糖鎖構造解析などの手法を用いて測定することができる。  [0055] In the method for suppressing the amount of N-glycolylneuraminic acid of the present invention, the method for culturing cells capable of producing a glycoprotein composition can be performed as described above. In addition, the amount of N-glycolylneuraminic acid bound to the glycoprotein composition was determined using the quantitative method and the two-dimensional sugar chain mapping method, as with the amount of sialic acid added to the glycoprotein composition. It can be measured using techniques such as sugar chain structure analysis.
[0056] 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ない。  [0056] 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
[0057] アンチトロンビンを生産する CHO細胞の 250mL三角フラスコを用いたフエドバッチ 培養における N—ァセチルノイラミン酸を培養開始時に添加することによる効果 アンチトロンビンを生産する CHO細胞株（FERM— BP8472)を用いて、以下の 2 50mL三角フラスコでのフエドバッチ培養を行い、 N—ァセチルノイラミン酸を培養開 始時に添加することによる効果を評価した。  [0057] Effect of adding N-acetylneuraminic acid at the start of culture in a fed-batch culture using 250 mL Erlenmeyer flasks of CHO cells that produce antithrombin A CHO cell line that produces antithrombin (FERM-BP8472) The following batch culture was performed in 250 mL Erlenmeyer flasks, and the effects of adding N-acetylneuraminic acid at the start of the culture were evaluated.
[0058] 本培養までの拡大培養用培地には、 EX— CELL™ 302培地 [シグマ.アルドリツ チ 'ファインケミカル（以下、 SAFCと称す）社製]に、メトトレキセー HSAFC社製、以 下、 MTXと称す） 500nmol/Lおよび L—グルタミン（和光純薬工業社製） 0· 875g /Lを添加した培地を用いた。 125mL、 250mLまたは lOOOmL容量の三角フラス コ（コーユング社製）に約 10〜30%量の培地を入れ、 3 X 105細胞/ mLとなるように 細胞懸濁液を播種した。その後、 35°Cで 3日間または 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の継代培養を行った。 [0058] The medium for expansion until the main culture is EX-CELL ™ 302 medium (Sigma. Aldrich 'Fine Chemical (hereinafter referred to as SAFC)), Methotrexe HSAFC (hereinafter referred to as MTX). A medium supplemented with 500 nmol / L and L-glutamine (manufactured by Wako Pure Chemical Industries, Ltd.) 0 · 875 g / L was used. About 10-30% of the medium was placed in a 125 mL, 250 mL, or lOOOOmL volume of triangular flask (manufactured by Cojung), and the cell suspension was seeded at 3 × 10 5 cells / mL. Thereafter, the cells were cultured at 35 ° C. for 3 or 4 days, and subculture was performed several times until the number of cells necessary for seeding of the main culture was obtained.
[0059] 本培養用の培地として、上記の拡大培養用培地に終濃度が lmmol/L、 5mmol し、 lOmmol し、 20mmol し、 40mmol し、 60mmol し、 80mmol Lまた は 1 OOmmol/Lとなるように N -ァセチルノイラミン酸（マルキンバイオ社製）をそれ ぞれ添加し、浸透圧を 330mOsm/kg、 pH7. 1前後に調整した培地をあらかじめ 調製した。また、コントロールとして上記シアル酸類（N ァセチルノイラミン酸）無添 加の培地も調製した。これらの N ァセチルノイラミン酸濃度が異なる本培養培地 50 mLを 250mL容量の三角フラスコ（コーユング社製）にそれぞれ注いだ。各々該本培 養培地に拡大培養で得た細胞を 3. 0 X 105細胞/ mLとなるように播種し、その後、 35°C、 100rpm、 5% CO環境下で 14日間の培養を行った。培養 3日目以降はフィ [0059] As a medium for main culture, the final concentration in the medium for expansion culture is 1 mmol / L, 5 mmol. N-acetyl-neuraminic acid (manufactured by Malkin Bio Inc.) was added to give an osmotic pressure of 330 mOsm to 10 mmol, 20 mmol, 40 mmol, 60 mmol, 60 mmol, 80 mmol L or 1 OO mmol / L, respectively. A medium adjusted to around / kg, pH 7.1 was prepared in advance. As a control, a medium without the above sialic acid (N-acetylneuraminic acid) was also prepared. 50 mL of these main culture media having different N-acetylneuraminic acid concentrations were poured into 250 mL Erlenmeyer flasks (manufactured by Cojung). The cells obtained by expansion culture are seeded at 3.0 × 10 5 cells / mL in the main culture medium, and then cultured for 14 days in a 35 ° C., 100 rpm, 5% CO environment. It was. After the third day of culture
2  2
ードとして 200g/Lの L グルコース（和光純薬工業社製)水溶液を培養液中の L— グルコース濃度が約 4g/Lとなるように添加した。  A 200 g / L aqueous solution of L glucose (manufactured by Wako Pure Chemical Industries, Ltd.) was added as a mode so that the L-glucose concentration in the culture solution was about 4 g / L.
[0060] 培養液を 3日目、 5日目、 7日目、 9日目、 11日目および 14日目に採取し、生細胞 密度（細胞/ mL)、生存率（％)および蛋白質生産濃度 (mg/L)をそれぞれ測定し た。また、培養終了時 (培養 14日目）の培養液を取得し、該培養液に生産された糖 蛋白質に結合している糖鎖末端のシアル酸付加量および N グリコリルノィラミン酸 付加量を測定した。 [0060] Cultures were collected on days 3, 5, 7, 9, 11, and 14 to determine viable cell density (cells / mL), viability (%), and protein production. Each concentration (mg / L) was measured. Also, a culture solution at the end of the culture (the 14th day of culture) is obtained, and the amount of sialic acid added at the end of the sugar chain and the amount of N-glycolylneuraminic acid added to the glycoprotein produced in the culture solution are measured. It was measured.
[0061] 測定方法については、生細胞密度および生存率は 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、蛋白質生産濃度は Method— Pair Antib ody Set for ELISA of human Antithronbin antigen ATIII) (ァフィニ ティ バイオロジカノレズ社製）を用いた ELISA法をそれぞれ用いて行った。 N ァセ チルノイラミン酸および N グリコリルノィラミン酸の付加量はそれぞれ 1 , 2—ジァミノ —4, 5—メチレンジォキシ ベンゼン（DMB)を用いた蛍光標識化（TaKaRaシアル 酸蛍光標識用試薬キット、タカラバイオ社製） HPLC法を用いて測定した。シァノレ 酸付加量はこれらの和として求めた。  [0061] Regarding the measurement method, the viable cell density and viability were 0.4% trypan blue solution (manufactured by Invitrogen), the dye exclusion method, and the protein production concentration was Method— Pair Antibody Set for ELISA of human Antithronbin. ELISA was performed using antigen ATIII) (manufactured by Affinity Biologics). The amount of N-acetylneuraminic acid and N-glycolylneuraminic acid added was 1,2-diamino-4,5-methylenedioxybenzene (DMB) for fluorescent labeling (TaKaRa sialic acid fluorescent labeling reagent kit, Takara Bio (Measured by HPLC). The amount of cyanole acid added was determined as the sum of these.
[0062] また、下記の式 2を用いて累積生細胞密度を、下記の式 3を用いて単位時間当たり に単位細胞が生産する蛋白量速度を表す比生産速度（SPR)を、下記の式 4を用い て相対シアル酸付加率（％)および下記の式 5を用いて相対 N グリコリルノィラミン 酸付加率（％)をそれぞれ算出した。  [0062] Further, the following formula 2 is used to calculate the cumulative viable cell density, and the following formula 3 is used to express the specific production rate (SPR) representing the protein amount rate produced by the unit cell per unit time. 4 was used to calculate the relative sialic acid addition rate (%) and the following formula 5 was used to calculate the relative N-glycolylneuraminic acid addition rate (%).
(式 2) 累積生細胞密度 [ (細胞/ mL) X日] =経過時間の始めと終わりの生細胞密度の 和（細胞/ mL) ÷ 2 X経過時間（日 )の総和 (Formula 2) Cumulative viable cell density [(cells / mL) X days] = sum of viable cell densities at the beginning and end of elapsed time (cells / mL) ÷ 2 X total elapsed time (days)
(式 3) (Formula 3)
SPR g/ ( 106細胞 X日）] =蛋白質生産濃度（ μ g/mL) ÷累積生細胞密度 [ (106細胞/111 X日] SPR g / (10 6 cells x day)] = protein production concentration (μg / mL) ÷ cumulative viable cell density [(10 6 cells / 111 x day)
(式 4) (Formula 4)
相対シアル酸付加率（％) =シアル酸類添加培養時における糖蛋白質中のシアル 酸付加量 (数/分子） ÷シアル酸類無添加培養時における糖蛋白質中のシアル酸 付加量 (数/分子） X 100  Relative sialic acid addition rate (%) = Sialic acid addition amount in glycoprotein during sialic acid addition culture (number / molecule) ÷ Sialic acid addition amount in glycoprotein during sialic acid addition-free culture (number / molecule) X 100
(式 5) (Formula 5)
相対 N—ダリコリルノイラミン酸付加率（％ ) =シアル酸類添加培養時における糖蛋 白質中の N—ダリコリルノィラミン酸付加量 (数/分子） ÷シアル酸類無添加培養時 における糖蛋白質中の N—ダリコリルノィラミン酸付加量 (数/分子） X 100  Relative N-darlicolylneuraminic acid addition rate (%) = N-darlicolylneuraminic acid addition amount (number / molecule) in glycoprotein during sialic acid-added culture ÷ glycoprotein in sialic acid-free culture N—Daricolylneuraminic acid addition amount (number / molecule) X 100
その結果を図 1〜図 3に示す。図 1に示したように、培養 14日目における糖蛋白質 に結合している糖鎖へのシアル酸付加量は培地に添加した N—ァセチルノイラミン 酸添加濃度に依存して増大し、 N—ァセチルノイラミン酸無添加培養時を 100%とし て比較した相対シアル酸付加率は多いもので 190%を越えた。また、図 2に示したよ うに、最も高濃度である 100mmol/Lの N—ァセチルノイラミン酸を添加した培地を 用いた細胞培養においても、培養 14日目までの生細胞密度および生存率の著しい 低下は認められず、最大到達生細胞密度は培養 8日目に 4. 4 X 106細胞/ mL以上 に達した。また、図 3に示したように、 14日間の培養終了時において、グラフの傾きで 示される SPRは N—ァセチルノイラミン酸添加濃度による影響は認められず、生産さ れた蛋白質生産濃度は約 160〜200mg/Lと、 N—ァセチルノイラミン酸無添加培 養時とほぼ同量であった。 20mmol/Lの N—ァセチルノイラミン酸を添加した培地 を用いた細胞培養の培養 14日目における、 N—ァセチルノイラミン酸無添加培養時 を 100%として比較した相対 N—グリコリルノィラミン酸付加率は 36%であり、 N—ダリ コリルノイラミン酸付加量の抑制が認められ、高品質のアンチトロンビン組成物を得る ことができた。 [0063] また、上述の方法で得られたシアル酸付加量が高いアンチトロンビンをゥサギ体内 へ導入した結果、血中半減期は、シアル酸無添加で培養して得られたアンチトロンビ ンに比べ、延長されることが示された。 The results are shown in Figs. As shown in Fig. 1, the amount of sialic acid added to the sugar chain bound to the glycoprotein on the 14th day of culture increases depending on the concentration of N-acetylneuraminic acid added to the medium. -The relative sialic acid addition rate compared to 100% in the culture without acetylylneuraminic acid was over 190%. In addition, as shown in FIG. 2, even in cell culture using a medium supplemented with 100 mmol / L N-acetylneuraminic acid, which is the highest concentration, the viable cell density and viability up to the 14th day of the culture were increased. There was no significant decrease, and the maximum viable cell density reached 4.4 × 10 6 cells / mL or more on the 8th day of culture. In addition, as shown in Fig. 3, at the end of 14 days of culture, the SPR indicated by the slope of the graph was not affected by the N-acetylneuraminic acid addition concentration, and the protein production concentration produced was The amount was about 160 to 200 mg / L, which was almost the same as that in the culture without addition of N-acetylneuraminic acid. Cultivation of cell culture using medium supplemented with 20 mmol / L of N-acetylneuraminic acid Relative N-glycolylneurine compared on the 14th day with no addition of N-acetylneuraminic acid as 100% The laminic acid addition rate was 36%, and the amount of N-dalicolylneuraminic acid addition was suppressed, and a high-quality antithrombin composition could be obtained. [0063] Further, as a result of introducing antithrombin having a high sialic acid addition amount obtained by the above-mentioned method into a rabbit, the blood half-life is compared with that obtained by culturing without sialic acid. It was shown to be extended.
実施例 2  Example 2
[0064] アンチトロンビンを生産する CHO細胞の 250mL三角フラスコを用いたフエドバッチ 培養におけるシアル酸ダイマーまたはコロミン酸を培養開始時に添加することによる 効果  [0064] Effect of adding sialic acid dimer or colominic acid at the start of culture in fed-batch culture using 250 mL Erlenmeyer flask of CHO cells producing antithrombin
アンチトロンビンを生産する CHO細胞株（FERM— BP8472)を用いて、以下の 2 50mL三角フラスコでのフエドバッチ培養を行い、シアル酸ダイマーまたはコロミン酸 を培養開始時に添加することによる効果を評価した。  Using the CHO cell line that produces antithrombin (FERM-BP8472), the following batch culture was performed in a 250 mL Erlenmeyer flask, and the effect of adding sialic acid dimer or colominic acid at the start of the culture was evaluated.
[0065] 本培養までの拡大培養は、実施例 1と同様の方法を用いた。 [0065] The same method as in Example 1 was used for the expansion until the main culture.
本培養用の培地として、実施例 1の拡大培養用培地にさらに、終濃度 0. 5mmol/ Lめるレヽは 5mmolZLのシァノレ酸タ マー [N— Ac etylneuraminic Acid, dime r ( a , 2→8) (ナカライテスタ社製）]、または終濃度 0. 05mmol/Lあるいは 0. 5m mol/Lのコロミン酸（ナカライテスタ社製）を添加し、浸透圧を 330mOsm/kg、 pH 7. 1前後に調整した培地をあらかじめ調製した。また、終濃度 5mmol/Lまたは 10 mmol/Lの N—ァセチルノイラミン酸 (協和発酵工業社製）を添加した培地およびコ ントロールとして上記シアル酸類（シアル酸ダイマー、コロミン酸または N—ァセチル ノィラミン酸）無添加の培地も調製した。各々の培地 50mLを 250mL三角フラスコ（コ 一ユング社製）にそれぞれ注いだ。該本培養培地の各々に拡大培養で調製された 細胞を 3. 0 X 105細胞/ mLとなるように播種し、その後、 35°C、 100rpm、 5% CO As the medium for main culture, the final concentration of 0.5 mmol / L was added to the medium for expansion culture of Example 1 with a 5 mmol ZL cyanoleic acid tag [N-Acylyluraminic Acid, dimer (a, 2 → 8 ) (Manufactured by Nacalai Testa)], or a final concentration of 0.05 mmol / L or 0.5 mmol / L of colominic acid (Nacalai Testa) is added to bring the osmotic pressure to around 330 mOsm / kg, pH 7.1 A conditioned medium was prepared in advance. In addition, sialic acids (sialic acid dimer, colominic acid or N-acetylylneuramin) can be used as a medium and control with N-acetylneuraminic acid (manufactured by Kyowa Hakko Kogyo Co., Ltd.) with a final concentration of 5 mmol / L or 10 mmol / L. An acid-free medium was also prepared. 50 mL of each medium was poured into a 250 mL Erlenmeyer flask (manufactured by Koyung). Cells prepared by expansion culture are seeded at 3.0 × 10 5 cells / mL in each of the main culture media, and then 35 ° C, 100 rpm, 5% CO.
2 環境下で 14日間の培養を行った。培養 3日目以降はフィードとして 200g/Lの L— グルコース（和光純薬工業社製)水溶液を培養液中の L—グルコース濃度が約 4g/ Lとなるように添加した。  2 Cultured for 14 days in an environment. After the third day of culture, 200 g / L L-glucose (manufactured by Wako Pure Chemical Industries, Ltd.) aqueous solution was added as a feed so that the L-glucose concentration in the culture solution was about 4 g / L.
[0066] 培養終了時 (培養 14日目）の培養液を取得し、該培養液から生産された糖蛋白質 に結合している糖鎖末端のシアル酸付加量および N—グリコリルノィラミン酸付加量 を測定した。  [0066] A culture solution at the end of the culture (the 14th day of culture) is obtained, and the amount of sialic acid added to the end of the sugar chain and N-glycolylneuraminic acid added to the glycoprotein produced from the culture solution The amount was measured.
測定方法につ!/、ては、実施例 1に記載された方法と同様の方法で行った。 また、実施例 1記載の式 4を用いて相対シアル酸付加率（％)および式 5を用いて相 対 N—グリコリルノィラミン酸付加率（％)をそれぞれ算出した。 Regarding the measurement method, the same method as that described in Example 1 was used. In addition, relative sialic acid addition rate (%) was calculated using Formula 4 described in Example 1, and relative N-glycolylneuraminic acid addition rate (%) was calculated using Formula 5.
[0067] 図 4に N—ァセチルノイラミン酸を添加して培養した場合の結果を、図 5にシアル酸 ダイマーを添加して培養した場合の結果を、図 6にコロミン酸を添加して培養した場 合の結果をそれぞれ示す。シアル酸ダイマーおよびコロミン酸を添加した培地を用い た細胞培養でも、培養 14日目における糖蛋白質に結合している糖鎖へのシアル酸 付加量が N—ァセチルノイラミン酸を添加した場合と同等、またはそれ以上に上昇し ていることが判明した。いずれの物質を用いた場合も、添加濃度依存的に相対シァ ノレ酸付加率が向上していた。また、 5mmol/Lのシアル酸ダイマーまたは 0· 5mmo 1/Lのコロミン酸を添加した培地を用いた細胞培養の培養 14日目における、シアル 酸類無添加培養時を 100%として比較した相対 N—グリコリルノィラミン酸付加率は それぞれ 55%、 45%であり、 N—グリコリルノィラミン酸付加量の抑制が認められた。 実施例 3 [0067] Fig. 4 shows the results when cultured with N-acetylneuraminic acid added, Fig. 5 shows the results when cultured with sialic acid dimer added, and Fig. 6 shows the results when colominic acid is added. The results when cultured are shown. Even in cell culture using a medium supplemented with sialic acid dimer and colominic acid, the amount of sialic acid added to the sugar chain bound to the glycoprotein on the 14th day of culture was the same as when N-acetylethylneuraminic acid was added. It has been found that it has risen to the same or higher level. In either case, the relative cyanoleic acid addition rate was improved depending on the addition concentration. In addition, relative N— compared with 100% of sialic acid-free culture on the 14th day of cell culture using 5 mmol / L sialic acid dimer or medium supplemented with 0.5 mmo 1 / L colominic acid. Glycolylneuraminic acid addition rates were 55% and 45%, respectively, and suppression of N-glycolylneuraminic acid addition was observed. Example 3
[0068] アンチトロンビンを生産する CHO細胞の 250mL三角フラスコを用いたフエドバッチ 培養における α 2, 3—シァリルラタトースまたは α 2, 6—シァリルラタトースを培養開 始時に添加することによる効果  [0068] Effect of adding α 2,3-sialyl ratatose or α 2,6-sialyl ratatose at the start of culture in a fed-batch culture using 250 mL Erlenmeyer flasks of CHO cells producing antithrombin
アンチトロンビンを生産する CHO細胞株（FERM— BP8472)を用いて、以下の 2 50mL三角フラスコでのフエドバッチ培養を行い、 « 2, 3—シァリルラタトースまたは a 2, 6—シァリルラタトースを培養開始時に添加することによる効果を調べた。  Using a CHO cell line that produces antithrombin (FERM—BP8472), perform batch batch culture in the following 250 mL Erlenmeyer flasks, and use «2,3-sialyl ratatose or a 2,6-sialyl ratatose The effect of adding at the start of the culture was examined.
[0069] 本培養までの拡大培養は、実施例 1と同様の方法を用いた。  [0069] The same method as in Example 1 was used for the expansion until the main culture.
本培養用の培地として、実施例 1の拡大培養用培地に、 α 2, 3—シァリルラタトー ス（協和発酵工業社製)または《 2, 6—シァリルラ外ース（協和発酵工業社製)をそ れぞれ終濃度 4mmol/Lまたは 20mmol/Lとなるように添加し、浸透圧を 330mO sm/kg、 pH7. 1前後に調整した培地を調製した。また、終濃度 4mmol/Lまたは 2 Ommol/Lの N—ァセチルノイラミン酸 (協和発酵工業社製）を添加した培地および コントロールとして上記シアル酸類（α 2, 3—シァリルラタトース、 α 2, 6—シァリルラ クトースまたは Ν—ァセチルノイラミン酸）無添加の培地も調製した。各々の培地 50m Lを 250mL三角フラスコ（コーユング社製）に注いだ。該本培養培地の各々に拡大 培養で調製した細胞を 3. 0 X 105細胞/ mLとなるように播種し、その後 35°C、 100r pm、 5% CO環境下で 14日間培養を行った。培養 3日目以降はフィードとして 200g As the medium for main culture, α 2,3-Sialyl ratose (manufactured by Kyowa Hakko Kogyo Co., Ltd.) or << 2,6-Sialyl lathose (manufactured by Kyowa Hakko Kogyo Co., Ltd.) is used as the medium for expansion culture of Example 1. Each medium was added so that the final concentration was 4 mmol / L or 20 mmol / L, and the osmotic pressure was adjusted to around 330 mO sm / kg and pH 7.1. In addition, a medium supplemented with 4 mmol / L or 2 Ommol / L of N-acetylneuraminic acid (manufactured by Kyowa Hakko Kogyo Co., Ltd.) and the above sialic acids (α 2,3-sialylatatos, α 2 , 6-sialyllactose or ァ -acetylneuraminic acid) -free medium was also prepared. 50 mL of each medium was poured into a 250 mL Erlenmeyer flask (Coujung). Expand to each of the main culture media Cells prepared by culture were seeded at 3.0 × 10 5 cells / mL, and then cultured in a 35 ° C., 100 rpm, 5% CO environment for 14 days. 200g feed after 3rd culture
2  2
/Lの L ダルコース（和光純薬工業社製)水溶液を培養液中の L ダルコース濃度 が約 4g/Lとなるように添加した。  / L L-dulose (manufactured by Wako Pure Chemical Industries, Ltd.) aqueous solution was added so that the L-darcose concentration in the culture solution was about 4 g / L.
[0070] 培養液を 3日目、 5日目、 7日目、 9日目、 11日目および 14日目に採取し、生細胞 密度（細胞/ mL)、生存率（％)および蛋白質生産濃度 (mg/L)をそれぞれ測定し た。また、培養終了時 (培養 14日目）の培養液を取得し、該培養液から生産された糖 蛋白質に結合している糖鎖末端のシアル酸付加量および N グリコリルノィラミン酸 付加量を測定した。 [0070] Cultures were collected on days 3, 5, 7, 9, 11, and 14 to determine viable cell density (cells / mL), viability (%), and protein production. Each concentration (mg / L) was measured. In addition, a culture solution at the end of the culture (the 14th day of culture) is obtained, and the amount of sialic acid added at the end of the sugar chain and the amount of N-glycolylneuraminic acid added to the glycoprotein produced from the culture solution are measured. It was measured.
[0071] シアル酸付加量および N グリコリルノィラミン酸付加量の測定方法については、 実施例 1と同様の方法で行った。培養液中のシァリダーゼ活性は文献 [Glycobiolog y, 3, 455 -463 (1993) ]記載の方法に基づいて測定した。  [0071] The methods for measuring the sialic acid addition amount and N-glycolylneuraminic acid addition amount were the same as in Example 1. The sialidase activity in the culture broth was measured based on the method described in the literature [Glycobiology, 3, 455-463 (1993)].
また、実施例 1記載の式 4を用いて相対シアル酸付加率（％)および式 5を用いて相 対 N グリコリルノィラミン酸付加率（％)をそれぞれ算出した。  Moreover, the relative sialic acid addition rate (%) was calculated using Formula 4 described in Example 1, and the relative N-glycolylneuraminic acid addition rate (%) was calculated using Formula 5.
[0072] その結果を図 7に示す。培養 14日目における相対シアル酸付加率は α 2, 3 シァ リルラタトースまたは α 2, 6 シァリルラタトース添加培地で濃度依存的に上昇して おり、 20mmol/Lの α 2、 3 シァリルラタトース添加では最大で 180%を上回った 。また、 20mmol/Lの α 2, 3 シァリノレラタトース、また (ま 20mmol/Lの α 2, 6— シァリルラタトースを添加した培地を用いた細胞培養の培養 14日目における、シアル 酸類無添加培養時を 100%として比較した相対 Ν グリコリルノィラミン酸付加率は それぞれ 27%、 45%であり、 Ν グリコリルノィラミン酸付加量の抑制が認められた。  The results are shown in FIG. The relative sialic acid addition rate on the 14th day of culture increased in a concentration-dependent manner in the medium supplemented with α2,3 sialyllatatose or α2,6 sialyllatatose, and 20 mmol / L of α2,3 sialyllatatose The maximum addition exceeded 180%. In addition, sialic acids on the 14th day of cell culture using 20 mmol / L α 2,3 sialino leratotase or medium supplemented with 20 mmol / L α 2,6- sialyl latatose The relative addition rate of glycolylneuraminic acid was 27% and 45%, respectively, compared with the non-additive culture as 100%.
[0073] 培養 14日目におけるシァリダーゼ活性はシアル酸無添加のコントロール培養では 0. 04U/mLであったが、 20mmol/Lの α 2, 3 シァリルラタトース添加培地では 約 0. 01U/mLに抑制されていた。  [0073] The sialidase activity on day 14 of culture was 0.04 U / mL in the control culture without sialic acid, but about 0.01 U / mL in the 20 mmol / L α 2,3 sialyl ratatoose-added medium. Was suppressed.
また、 20mmol/Lの α 2, 3 シァリルラタトースまたは 20mmol/Lの α 2, 6 シ ァリルラタトースを培地に添加した培養において、生細胞密度、生存率および蛋白質 生産濃度は、 Ν ァセチルノイラミン酸を添加した培地での培養と大きな差はなかつ た。 実施例 4 In addition, in a culture in which 20 mmol / L α 2,3 sialyl latatose or 20 mmol / L α 2,6 sialyl latatose was added to the medium, the viable cell density, viability, and protein production concentration were There was no significant difference from culture in a medium supplemented with acid. Example 4
[0074] アンチトロンビンを生産する CHO細胞の 250mL三角フラスコを用いたフエドバッチ 培養における N—ァセチルノイラミン酸を対数増殖期または静止期に添加することに よる効果  [0074] Effects of adding N-acetylneuraminic acid in the logarithmic growth phase or stationary phase in a fed-batch culture using 250 mL Erlenmeyer flasks of CHO cells producing antithrombin
アンチトロンビンを生産する CHO細胞株（FERM— BP8472)を用いて、以下の 2 50mL三角フラスコでのフエドバッチ培養を行い、 N—ァセチルノイラミン酸を対数増 殖期および静止期に添加することによる効果を調べた。  Using a CHO cell line (FERM-BP8472) that produces antithrombin, fed batch culture in the following 250 mL Erlenmeyer flask and adding N-acetylneuraminic acid to the logarithmic growth phase and stationary phase The effect was investigated.
[0075] 本培養までの拡大培養は、実施例 1と同様の方法を用いた。 [0075] For the expansion culture up to the main culture, the same method as in Example 1 was used.
本培養用の培地として、実施例 1の拡大培養用培地を用い、浸透圧を 330mOsm /kg、 pH7. 1前後に調製して 250mL容量の三角フラスコ（コーユング社製）に約 5 OmLの培地を入れ、 3 X 105細胞/ mLとなるように細胞懸濁液を播種し、その後 35 °C、 100rpm、 5% CO環境下で 14日間培養を行った。 As the medium for main culture, the medium for expansion culture of Example 1 was used. The osmotic pressure was adjusted to 330 mOsm / kg and pH 7.1, and about 5 OmL of medium was added to a 250 mL Erlenmeyer flask (manufactured by Coung). Then, the cell suspension was seeded at 3 × 10 5 cells / mL, and then cultured in a 35 ° C., 100 rpm, 5% CO environment for 14 days.
2  2
[0076] 培養 3日目以降はフィードとして 200g/Lの L—グノレコース（和光純薬工業社製） 水溶液を培養液中の L—グルコース濃度が約 4g/Lとなるように添加した。  [0076] After the third day of culture, 200 g / L of L-gnolecose (manufactured by Wako Pure Chemical Industries, Ltd.) as an aqueous solution was added as a feed so that the L-glucose concentration in the culture solution was about 4 g / L.
さらに、培養 5日目、 8日目、 9日目、 10日目、 11日目、 12日目または 13日目に培 養液に N—ァセチルノイラミン酸 (協和発酵工業株式会社製）の濃縮液を終濃度 30 mmol/Lとなるように添加した。また、コントロールとして上記シアル酸類（N—ァセ チルノイラミン酸）無添加での培養も行った。  Furthermore, N-acetylmethylneuraminic acid (manufactured by Kyowa Hakko Kogyo Co., Ltd.) was added to the culture solution on the 5th, 8th, 9th, 10th, 11th, 12th or 13th day of culture. Was added to a final concentration of 30 mmol / L. Further, as a control, culture without the above sialic acid (N-acetylneuraminic acid) was also performed.
[0077] 培養液を 3日目、 5日目、 8日目、 11日目および 14日目に採取し、生細胞密度（細 胞/ mL)および生存率（％)をそれぞれ測定した。また、培養終了時 (培養 14日目） の培養液を取得し、該培養液から生産された糖蛋白質に結合している糖鎖末端のシ アル酸付加量を測定した。  [0077] Culture media were collected on days 3, 5, 8, 11, and 14, and the viable cell density (cells / mL) and viability (%) were measured, respectively. In addition, a culture solution at the end of the culture (culture day 14) was obtained, and the amount of sialic acid added at the sugar chain end bound to the glycoprotein produced from the culture solution was measured.
測定方法につ!/、ては、実施例 1に記載された方法と同様の方法で行った。  Regarding the measurement method, the same method as that described in Example 1 was used.
[0078] また、実施例 1記載の式 4を用いて相対シアル酸付加率（％)を算出した。  In addition, the relative sialic acid addition rate (%) was calculated using Formula 4 described in Example 1.
その結果を図 8および図 9に示す。図 8に示したように、 N—ァセチルノイラミン酸の 添加時期の違いが相対シアル酸付加率に顕著に影響した。培養 11日目までは N— ァセチルノイラミン酸添加によるシアル酸付加量の上昇効果が観察された。一方で、 培養 12日目での N—ァセチルノイラミン酸添加では、相対シアル酸付加率が 11日 目までの添加と比べて低下した。図 9に示したように、培養 12日目以降は生細胞密 度が急速に低下する傾向にあることから、培養 11日目までを静止期と捉えることが出 来る。即ち、生産する糖蛋白質中のシアル酸の付加率を増加させるためには、静止 期（この場合培養 11日目まで）までにシアル酸を添加する必要があることが明らかに なった。また、シアル酸の添加時期に関わらず、培養細胞の生存率および生細胞密 度に大きな相違は観察されなかった。 The results are shown in Figs. As shown in Fig. 8, the difference in the addition timing of N-acetylneuraminic acid significantly affected the relative sialic acid addition rate. Up to the 11th day of culture, the effect of increasing the amount of sialic acid added by adding N-acetylneuraminic acid was observed. On the other hand, when N-acetylneuraminic acid was added on the 12th day of culture, the relative sialic acid addition rate was 11 days. Compared to the addition up to the eyes. As shown in Fig. 9, the viable cell density tends to decrease rapidly after the 12th day of culture, so that the 11th day of culture can be regarded as the stationary phase. In other words, in order to increase the addition rate of sialic acid in the glycoprotein to be produced, it became clear that sialic acid had to be added by the stationary phase (in this case, until the 11th day of culture). Regardless of the time of addition of sialic acid, no significant difference was observed in the viability and viability of cultured cells.
実施例 5  Example 5
[0079] アンチトロンビンを生産する CHO細胞の 250mL三角フラスコを用いた N ァセチ ルノイラミン酸を培養開始時に添加したフエドバツチ培養における浸透圧による効果 アンチトロンビンを生産する CHO細胞株（FERM— BP8472)を用いて、以下の N ーァセチルノイラミン酸を培養開始時に添加した 250mL三角フラスコでのフエドバッ チ培養を行い、浸透圧による効果を調べた。  [0079] Effect of osmotic pressure on Fuedbachi culture in which N-acetylneuraminic acid was added at the start of culture using 250 mL Erlenmeyer flask of CHO cells producing antithrombin Using CHO cell line (FERM-BP8472) producing antithrombin Then, fed-batch culture was performed in a 250 mL Erlenmeyer flask to which the following N-acetylmethylneuraminic acid was added at the start of the culture, and the effect of osmotic pressure was examined.
[0080] 本培養までの拡大培養は、実施例 1と同様の方法を用いた。  [0080] The same method as in Example 1 was used for the expansion culture until the main culture.
本培養用の培地として、実施例 1の拡大培養用培地にさらに N ァセチルノイラミン 酸（協和発酵工業株式会社製）を 20mmol/L、 40mmol/L、 60mmol/L、 80m mol/Lまたは lOOmmol/L添加し、浸透圧を 330mOsm/kg、 pH7. 1前後に調 製した培地と無調製の培地をそれぞれ調製し、 250mL容量の三角フラスコ（コ一二 ング社製）に約 50mLの培地を入れ、 3 X 105細胞/ mLとなるように細胞懸濁液を播 種し、その後 35°C、 100rpm、 5% CO環境下で 3日間培養を行った。 As a medium for main culture, N-acetylneuraminic acid (manufactured by Kyowa Hakko Kogyo Co., Ltd.) is further added to the expansion culture medium of Example 1 at 20 mmol / L, 40 mmol / L, 60 mmol / L, 80 mmol / L or lOOmmol. A medium prepared with osmotic pressure of 330 mOsm / kg and a pH of about 7.1 and a non-prepared medium were prepared, and about 50 mL of medium was added to a 250 mL Erlenmeyer flask (manufactured by Coning). Then, the cell suspension was seeded at 3 × 10 5 cells / mL, and then cultured in a 35 ° C., 100 rpm, 5% CO environment for 3 days.
2  2
[0081] 培養液を 3日目に採取し、生細胞密度（細胞/ mUを測定した。  [0081] The culture solution was collected on the third day, and the viable cell density (cells / mU was measured.
測定方法につ!/、ては、実施例 1に記載された方法と同様の方法で行った。 増殖速度 (h— を、培養 3日目の生細胞密度を用いて、以下の式 6より算出した。 (式 6)  Regarding the measurement method, the same method as that described in Example 1 was used. The growth rate (h— was calculated from the following formula 6 using the viable cell density on the third day of culture. (Formula 6)
増殖速度 (h— = (3日目生細胞密度の自然対数） （播種した細胞密度の自然対 数) /3 (日）/ 24 (h)  Growth rate (h— = (Natural logarithm of viable cell density on day 3) (Natural logarithm of seeded cell density) / 3 (day) / 24 (h)
その結果を図 10に示す。浸透圧無調整群では N ァセチルノイラミン酸の添加濃 度依存的に培養 3日目における細胞増殖速度は低下した力 浸透圧を調整すること で増殖阻害が回避された。 実施例 6 The results are shown in Fig. 10. In the osmotic pressure non-adjusted group, growth inhibition was avoided by adjusting the force osmotic pressure, which decreased the cell growth rate on the third day of culture depending on the concentration of N-acetylneuraminic acid. Example 6
[0082] アンチトロンビンを生産する CHO細胞の 2000mLバイオリアクターを用いたフエド ノ ッチ培養における N—ァセチルノイラミン酸を培養開始時に添加することによる効 果  [0082] Effect of adding N-acetylneuraminic acid at the start of culture in a fed-notch culture using 2000 mL bioreactor of CHO cells producing antithrombin
アンチトロンビンを生産する CHO細胞株（FERM— BP8472)を用いて、以下の 2 OOOmLバイオリアクターでのフエドバッチ培養を行い、 N—ァセチルノイラミン酸を培 養開始時に添加することによる効果を調べた。  Using the CHO cell line that produces antithrombin (FERM-BP8472), we performed fed-batch culture in the following 2 OOOmL bioreactor, and examined the effects of adding N-acetylneuraminic acid at the start of the culture. .
[0083] 本培養までの拡大培養は、実施例 1と同様の方法を用いた。  [0083] The same method as in Example 1 was used for the expansion until the main culture.
本培養用の培地として、実施例 1の拡大培養用培地に N—ァセチルノイラミン酸（ 協和発酵工業社製）を終濃度 20mmol/Lまたは 40mmol/Lとなるように添加して 培地を調製した。また、同時にコントロールとして上記シアル酸類（N—ァセチルノィ ラミン酸）無添加の培地も調製した。各々の培地 700mLを 2000mL容量バイオリア クタ一（エイブル社製）に注いだ。拡大培養で調製した細胞を 3. 0 105細胞/111しと なるように播種し、その後、 35°C、 85rpm、 pH7. 1環境下で 14日間培養を行った。 As a medium for main culture, add N-acetylneuraminic acid (manufactured by Kyowa Hakko Kogyo Co., Ltd.) to the expansion culture medium of Example 1 to a final concentration of 20 mmol / L or 40 mmol / L to prepare a medium. did. At the same time, a medium without the above-mentioned sialic acid (N-acetylaminolamic acid) was also prepared as a control. 700 mL of each medium was poured into a 2000 mL bioreactor (Able). Cells prepared by expansion culture were seeded at 3.0 10 5 cells / 111, and then cultured in an environment of 35 ° C., 85 rpm, pH 7.1 for 14 days.
[0084] フィード培地として、アミノ酸（L—ァラニン 0· 14g/L、 L—アルギニン一塩酸 0· 47 g/L、 Lーァスパラギン一水和物 0· 16g/L、 Lーァスパラギン酸 0· 17g/L、L— シスチン二塩酸 0· 51g/L、 L—グルタミン酸 0· 42g/L、 Lーグノレタミン 7. 3g/L、 グリシン 0· 1 7g/L、 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. 59g/L、： L—リジン一塩酸 0. 82g/:L、 L—メチォニン 0. 17g/:L、 L—フエニノレアラニン 0. 37g/L、： L—プロリン 0. 22g/L、： L—セリン 0. 2 4g八、： L—スレオニン 0. 53g/L、： L—卜リプ卜ファン 0. 09g/L,： L—チロシンニナ トリウム二水和物 0 · 58g/Lおよび Lーバリン 0· 53g/U、ビタミン（d—ビォチン 0· 073mg /し、 D—ノ ン卜テン酸カノレシゥム 0· 022g /し、塩ィ匕コリン 0· 022g /し、葉 酸 0. 022g/L、 myo—イノシトーノレ 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. 022g/ Lおよびシァノコバラミン 0· 073mg/U、リコンビナントヒトインスリン 0· 31g/L (以 上、 SAFC社製）、エタノールァミン（SAFC社製） 0· 025g/L、 2—メルカプトエタノ ール（SAFC社製） 0· 0098g/L、 Soy Hvdrolysate UF (SAFC社製） 8g/L、 亜セレン酸ナトリウム（SAFC社製） 16. 8 11 g/ コレステロール脂質濃縮溶液（25 0 X水溶液、インビトロジェン社製） 2mL/L、およびエチレンジァミン四酢酸第二鉄 ナトリウム塩（SAFC社製） 0· 05g/Lからなる溶液を、培養 3日目、 4日目、 5日目、 6 日目および 7日目に 1日 1回計 5回、それぞれ約 40mL添加した。さらにフィードとして 500g/Lの L—グルコース（和光純薬工業社製)水溶液を培養液中 L—グルコース 濃度が約 4g/Lとなるように、培養 3日目以降に毎日添加した。 [0084] As a feed medium, amino acids (L-alanin 0 · 14 g / L, L-arginine monohydrochloride 0 · 47 g / L, L-asparagine monohydrate 0 · 16 g / L, L-argasine acid 0 · 17 g / L , L-cystine dihydrochloride 0 · 51g / L, L-glutamic acid 0 · 42g / L, L-gnoretamine 7.3g / L, glycine 0 · 17g / L, L-histidine monohydrochloride dihydrate 0 · 24g / L L, L—isoleucine 0 · 59 g / L, L—leucine 0.59 g / L, L—lysine monohydrochloride 0.82 g /: L, L—methionine 0.17 g /: L, L—phenenolealanine 0. 37g / L,: L-proline 0.22g / L, L-serine 0.24g8, L-threonine 0.53g / L, L-Lip 卜 fan 0.09g / L, L- Tyrosine Ninatrium Dihydrate 0 · 58g / L and L-Valine 0 · 53g / U, Vitamin (d-biotin 0 · 073mg / d, D-non-succinic acid canoleum 0 · 022g / d, salt 匕 choline 0 · 022g / folic acid 0.022g / L, myo—Inosito Nore 0.040 g / L, niacinamide 0.022 g / L, pyridoxal hydrochloride 0.022 g / L, riboflavin 0.0022 g / L, thiamine hydrochloride 0.022 g / L and cyanobalamine 0 · 073 mg / U, recombinant human insulin 0 · 31g / L (SAFC), ethanolamine (SAFC) 0 · 25g / L, 2-mercaptoethanol (SAFC) 0 · 0098g / L, Soy Hvdrolysate UF (SAFC 8g / L, manufactured by Sodium selenite (SAFC) 16. 8 11 g / Cholesterol lipid concentrated solution (25 0 X aqueous solution, Invitrogen) 2 mL / L, and ethylenediammine ferric tetraacetate sodium salt (SAFC) 0 · 05g About 40 mL each of the solution consisting of / L was added once a day on the 3rd, 4th, 5th, 6th and 7th days of culture. Furthermore, 500 g / L L-glucose (manufactured by Wako Pure Chemical Industries, Ltd.) aqueous solution as a feed was added every day after the third day of culture so that the L-glucose concentration in the culture solution was about 4 g / L.
[0085] 培養液を 1日 1回採取し、生細胞密度（細胞/ mL)、生存率（％)および蛋白質生 産濃度（mg/L)をそれぞれ測定した。また、 8日目、 10日目、 12日目および 14日 目の培養液を取得し、該培養液に生産された糖蛋白質に結合している糖鎖末端の シアル酸付加量および N—グリコリルノィラミン酸付加量を測定した。  [0085] The culture broth was collected once a day, and the viable cell density (cells / mL), viability (%), and protein production concentration (mg / L) were measured. In addition, the culture solutions on the 8th, 10th, 12th and 14th days were obtained, and the amount of sialic acid added at the end of the sugar chain bound to the glycoprotein produced in the culture solution and N-glyco The amount of rilneuramic acid addition was measured.
測定方法につ!/、ては、実施例 1に記載された方法と同様の方法で行った。  Regarding the measurement method, the same method as that described in Example 1 was used.
[0086] また、実施例 1記載の式 4を用いて相対シアル酸付加率（％)を算出した。  [0086] The relative sialic acid addition rate (%) was calculated using Formula 4 described in Example 1.
さらに下記の式 7を用いてシアル酸中に占める N—グリコリルノィラミン酸相対含量（ %)を算出した。  Further, the relative content (%) of N-glycolylneuraminic acid in sialic acid was calculated using the following formula 7.
(式 7)  (Formula 7)
シアル酸中に占める N—ダリコリルノイラミン酸相対含量（％ ) =糖蛋白質中の N— グリコリルノィラミン酸付加量 (数/分子） ÷N—ァセチルノイラミン酸無添加培養 8日 目の糖蛋白質中の N—グリコリルノィラミン酸付加量 (数/分子） X 100  Relative content of N-dalicolyl neuraminic acid in sialic acid (%) = N-glycolylneuraminic acid addition in glycoprotein (number / molecule) ÷ N-acetylneuraminic acid-free culture day 8 Of N-glycolylneuraminic acid in some glycoproteins (number / molecule) X 100
その結果を図 11〜図 13に示す。図 11に示したように、糖蛋白質中の N—グリコリ ルノイラミン酸含量は、培養時に N—ァセチルノイラミン酸を添加することにより、添カロ 濃度に依存的な減少を示し、 N—ァセチルノイラミン酸を 40mmol/L添加した培養 の培養 14日目の N—グリコリルノィラミン酸の量は無添加群に対して 1/10となった 。また、図 12に示したように、培養 14日目における糖蛋白質に結合している糖鎖へ のシアル酸付加量は培地に添加した N—ァセチルノイラミン酸添加濃度に依存して 増大し、 N—ァセチルノイラミン酸無添加培養時を 100%として比較した相対シアル 酸付加率は多いもので 160%を越えた。また、図 13に示したように、最も高濃度であ る 40mmol/Lの N—ァセチルノイラミン酸を添加した培地を用いた細胞培養におい ても、培養 14日目までの生細胞密度および生存率の著しい低下は認められず、最 大到達生細胞密度は培養 10日目に 1. 0 X 107細胞/ mL以上に達した。 The results are shown in FIGS. As shown in Fig. 11, the content of N-glycolylneuraminic acid in glycoproteins decreased with the addition of N-acetylneuraminic acid during the culture, depending on the concentration of added caroline. The amount of N-glycolylneuraminic acid on the 14th day of culture in which 40 mmol / L of neuraminic acid was added was 1/10 of that in the non-added group. In addition, as shown in FIG. 12, the amount of sialic acid added to the sugar chain bound to the glycoprotein on the 14th day of culture increased depending on the concentration of N-acetylneuraminic acid added to the medium. The relative sialic acid addition rate was over 160% when compared to 100% in the culture without N-acetylneuraminic acid. In addition, as shown in FIG. 13, even in cell culture using a medium supplemented with the highest concentration of 40 mmol / L N-acetylneuraminic acid, the viable cell density up to the 14th day of culture and There was no significant decrease in survival rate. The maximum viable cell density reached 1.0 × 10 7 cells / mL or more on the 10th day of culture.
産業上の利用可能性 Industrial applicability
本発明により、シアル酸、シアル酸重合物およびシアル酸を含むオリゴ糖から選ば れる少なくとも 1種類の物質を培地に添加することを特徴とする糖蛋白質組成物の製 造方法、糖蛋白質組成物を生産する細胞の培養方法、該細胞より生産される糖蛋白 質組成物に結合するシアル酸の付加量を向上させる方法、および該細胞より生産さ れる糖蛋白質組成物に結合する N—グリコリルノィラミン酸の付加量を抑制する方法 が提供される。本発明により製造される糖蛋白質組成物は従来の糖蛋白質組成物よ りもシアル酸付加量が増加し、 N—グリコリルノィラミン酸含有量が抑制されることから 医薬品として有用である。  According to the present invention, a method for producing a glycoprotein composition, comprising adding at least one substance selected from sialic acid, a sialic acid polymer and an oligosaccharide containing sialic acid to a medium, and a glycoprotein composition A method for culturing cells to be produced, a method for improving the amount of sialic acid added to a glycoprotein composition produced from the cells, and an N-glycolylneune that binds to a glycoprotein composition produced from the cells A method for suppressing the amount of laminic acid added is provided. The glycoprotein composition produced according to the present invention is useful as a pharmaceutical because the amount of sialic acid added is higher than that of conventional glycoprotein compositions and the N-glycolylneuraminic acid content is suppressed.

Claims

請求の範囲 The scope of the claims
[1] 5〜200mmol/Lのシアル酸、 0· ；!〜 200mmol/Lのシアル酸重合物、；!〜 200 mmol/Lのシアル酸を含むオリゴ糖から選ばれる少なくとも 1種類の物質を添加した 培地中で細胞を培養し、培養物中に糖蛋白質組成物を生成蓄積させ、該培養物か ら該糖蛋白質組成物を採取することを特徴とする、糖蛋白質組成物の製造方法。  [1] 5 to 200 mmol / L sialic acid, 0 ·;! To 200 mmol / L sialic acid polymer;! To at least one substance selected from oligosaccharides containing 200 to 200 mmol / L sialic acid A method for producing a glycoprotein composition, comprising culturing cells in a medium, producing and accumulating the glycoprotein composition in the culture, and collecting the glycoprotein composition from the culture.
[2] シアル酸が N—ァセチルノイラミン酸である、請求項 1に記載の方法。  [2] The method according to claim 1, wherein the sialic acid is N-acetylneuraminic acid.
[3] シアル酸、シアル酸重合物およびシアル酸を含むオリゴ糖から選ばれる少なくとも 1 種類の物質を培養開始時、対数増殖期、もしくは静止期に培地へ添加する、請求項 1または 2に記載の方法。  [3] The method according to claim 1 or 2, wherein at least one substance selected from sialic acid, a sialic acid polymer, and an oligosaccharide containing sialic acid is added to the medium at the start of culture, logarithmic growth phase, or stationary phase. the method of.
[4] 細胞を浸透圧 250〜400mOsm/kgで培養する、請求項 1〜3のいずれ力、 1項に記 載の方法。  [4] The method according to any one of claims 1 to 3, wherein the cells are cultured at an osmotic pressure of 250 to 400 mOsm / kg.
[5] 細胞が動物細胞である、請求項 1〜4のいずれ力、 1項に記載の方法。  [5] The method according to any one of claims 1 to 4, wherein the cell is an animal cell.
[6] 動物細胞が、以下の ω〜α)からなる群から選ばれる細胞である、請求項 5に記載 の方法。  6. The method according to claim 5, wherein the animal cell is a cell selected from the group consisting of the following ω to α).
(a)チャイニーズノ、ムスター卵巣組織由来 CHO細胞；  (a) Chinese CHO, derived from Muster ovarian tissue;
(b)ラットミエ口一マ糸田月包ネ朱 YB2/3HL. P2. Gi l . 16Ag. 20糸田月包；  (b) Rat Mye Kouichi Ma Itohtsuki Parcel Zhu YB2 / 3HL. P2. Gi l. 16Ag.
(c)マウスミエローマ細胞株 NS0細胞；  (c) mouse myeloma cell line NS0 cells;
(d)マウスミエローマ細胞株 SP2/0— Agl4細胞；  (d) mouse myeloma cell line SP2 / 0—Agl4 cells;
(e)シリアンノ、ムスター腎臓組織由来 BHK細胞；  (e) Syrianno, Muster kidney tissue-derived BHK cells;
(f)抗体を産生するハイプリドーマ細胞；  (f) a hyperidoma cell producing the antibody;
(g)ヒト白血病細胞株ナマルバ細胞；  (g) human leukemia cell line Namalva cells;
(h)ヒト白血病細胞株 NM— F9細胞；  (h) human leukemia cell line NM-F9 cells;
(i)ヒト胚性網膜細胞株 PER. C6細胞；  (i) human embryonic retinal cell line PER. C6 cells;
(j)胚性幹細胞；  (j) embryonic stem cells;
(k)受精卵細胞；  (k) a fertilized egg cell;
(1)ヒ卜 HEK293細胞。  (1) Herbal HEK293 cells.
[7] 細胞が、糖蛋白質をコードする DNAが導入された細胞である、請求項;!〜 6のいず れか 1項に記載の方法。 [7] The method according to any one of [1] to [6] above, wherein the cell is a cell into which DNA encoding a glycoprotein has been introduced.
[8] 糖蛋白質がアンチトロンビンである、請求項 1〜7のいずれ力、 1項に記載の方法。 8. The method according to any one of claims 1 to 7, wherein the glycoprotein is antithrombin.
[9] 5〜200mmol/Lのシアル酸、 0. ；!〜 200mmol/Lのシアル酸重合物、；!〜 200 mmol/Lのシアル酸を含むオリゴ糖から選ばれる少なくとも 1種類の物質を添加した 培地中で培養することを特徴とする、糖蛋白質組成物を生産する細胞の培養方法。 [9] 5 to 200 mmol / L sialic acid, 0.;! To 200 mmol / L sialic acid polymer;! To at least one substance selected from oligosaccharides containing 200 to 200 mmol / L sialic acid A method for culturing cells producing a glycoprotein composition, comprising culturing in a medium.
[10] シアル酸が N—ァセチルノイラミン酸である、請求項 9に記載の方法。 [10] The method according to claim 9, wherein the sialic acid is N-acetylneuraminic acid.
[11] シアル酸、シアル酸重合物およびシアル酸を含むオリゴ糖から選ばれる少なくとも 1 種類の物質を培養開始時、対数増殖期、もしくは静止期に培地へ添加する、請求項[11] The at least one substance selected from sialic acid, sialic acid polymer and oligosaccharide containing sialic acid is added to the medium at the start of culture, in the logarithmic growth phase, or in the stationary phase.
9または 10に記載の方法。 The method according to 9 or 10.
[12] 細胞を浸透圧 250〜400mOsm/kgで培養する、請求項 9〜； 11のいずれか 1項に 記載の方法。 [12] The method according to any one of [9] to [11], wherein the cells are cultured at an osmotic pressure of 250 to 400 mOsm / kg.
[13] 細胞が動物細胞である、請求項 9〜； 12のいずれか 1項に記載の方法。  [13] The method according to any one of claims 9 to 12, wherein the cell is an animal cell.
[14] 動物細胞が、以下の（a)〜（l)からなる群から選ばれる細胞である、請求項 13に記載 の方法。  [14] The method according to [13], wherein the animal cell is a cell selected from the group consisting of the following (a) to (l).
(a)チャイニーズノ、ムスター卵巣組織由来 CHO細胞；  (a) Chinese CHO, derived from Muster ovarian tissue;
(b)ラットミエ口一マ糸田月包ネ朱 YB2/3HL. P2. Gi l . 16Ag. 20糸田月包；  (b) Rat Mye Kouichi Ma Itohtsuki Parcel Zhu YB2 / 3HL. P2. Gi l. 16Ag.
(c)マウスミエローマ細胞株 NS0細胞；  (c) mouse myeloma cell line NS0 cells;
(d)マウスミエローマ細胞株 SP2/0— Agl4細胞；  (d) mouse myeloma cell line SP2 / 0—Agl4 cells;
(e)シリアンノ、ムスター腎臓組織由来 BHK細胞；  (e) Syrianno, Muster kidney tissue-derived BHK cells;
(f)抗体を産生するハイプリドーマ細胞；  (f) a hyperidoma cell producing the antibody;
(g)ヒト白血病細胞株ナマルバ細胞；  (g) human leukemia cell line Namalva cells;
(h)ヒト白血病細胞株 NM— F9細胞；  (h) human leukemia cell line NM-F9 cells;
(i)ヒト胚性網膜細胞株 PER. C6細胞；  (i) human embryonic retinal cell line PER. C6 cells;
(j)胚性幹細胞；  (j) embryonic stem cells;
(k)受精卵細胞；  (k) a fertilized egg cell;
(1)ヒ卜 HEK293細胞。  (1) Herbal HEK293 cells.
[15] 細胞が、糖蛋白質をコードする DNAが導入された細胞である、請求項 9〜； 14のいず れか 1項に記載の方法。  [15] The method according to any one of [9] to [14], wherein the cell is a cell into which DNA encoding a glycoprotein has been introduced.
[16] 糖蛋白質がアンチトロンビンである、請求項 9〜； 15のいずれ力、 1項に記載の方法。 [16] The method according to any one of [9] to [15], wherein the glycoprotein is antithrombin.
[17] 5〜200mmol/Lのシアル酸、 0. ；!〜 200mmol/Lのシアル酸重合物、；!〜 200 mmol/Lのシアル酸を含むオリゴ糖から選ばれる少なくとも 1種類の物質を添加した 培地中で、糖蛋白質組成物を生産する能力を有する細胞を培養することを特徴とす る、該細胞より生産される糖蛋白質組成物に結合するシアル酸の付加量を向上させ る方法。 [17] 5 to 200 mmol / L sialic acid, 0.;! To 200 mmol / L sialic acid polymer;! To at least one substance selected from oligosaccharides containing 200 to 200 mmol / L sialic acid A method for improving the amount of sialic acid added to the glycoprotein composition produced from the cells, comprising culturing cells capable of producing the glycoprotein composition in a medium.
[18] シアル酸が N—ァセチルノイラミン酸である、請求項 17に記載の方法。  18. The method according to claim 17, wherein the sialic acid is N-acetylneuraminic acid.
[19] シアル酸、シアル酸重合物およびシアル酸を含むオリゴ糖から選ばれる少なくとも 1 種類の物質を培養開始時、対数増殖期、もしくは静止期に培地へ添加する、請求項 [19] The at least one substance selected from sialic acid, sialic acid polymer and oligosaccharide containing sialic acid is added to the medium at the start of culture, in the logarithmic growth phase, or in the stationary phase.
17または 18に記載の方法。 The method according to 17 or 18.
[20] 細胞を浸透圧 250〜400mOsm/kgで培養する、請求項 17〜19のいずれ力、 1項 に記載の方法。 [20] The method according to any one of claims 17 to 19, wherein the cells are cultured at an osmotic pressure of 250 to 400 mOsm / kg.
[21] 細胞が動物細胞である、請求項 17〜20のいずれか 1項に記載の方法。  [21] The method according to any one of claims 17 to 20, wherein the cell is an animal cell.
[22] 動物細胞が、以下の（a)〜（l)からなる群から選ばれる細胞である、請求項 21に記載 の方法。  [22] The method according to claim 21, wherein the animal cell is a cell selected from the group consisting of the following (a) to (l).
(a)チャイニーズノ、ムスター卵巣組織由来 CHO細胞；  (a) Chinese CHO, derived from Muster ovarian tissue;
(b)ラットミエ口一マ糸田月包ネ朱 YB2/3HL. P2. Gi l . 16Ag. 20糸田月包；  (b) Rat Mye Kouichi Ma Itohtsuki Parcel Zhu YB2 / 3HL. P2. Gi l. 16Ag.
(c)マウスミエローマ細胞株 NS0細胞；  (c) mouse myeloma cell line NS0 cells;
(d)マウスミエローマ細胞株 SP2/0— Agl4細胞；  (d) mouse myeloma cell line SP2 / 0—Agl4 cells;
(e)シリアンノ、ムスター腎臓組織由来 BHK細胞；  (e) Syrianno, Muster kidney tissue-derived BHK cells;
(f)抗体を産生するハイプリドーマ細胞；  (f) a hyperidoma cell producing the antibody;
(g)ヒト白血病細胞株ナマルバ細胞；  (g) human leukemia cell line Namalva cells;
(h)ヒト白血病細胞株 NM— F9細胞；  (h) human leukemia cell line NM-F9 cells;
(i)ヒト胚性網膜細胞株 PER. C6細胞；  (i) human embryonic retinal cell line PER. C6 cells;
(j)胚性幹細胞；  (j) embryonic stem cells;
(k)受精卵細胞；  (k) a fertilized egg cell;
(1)ヒ卜 HEK293細胞。  (1) Herbal HEK293 cells.
[23] 細胞が、糖蛋白質をコードする DNAが導入された細胞である、請求項 17〜22のい ずれか 1項に記載の方法。 23. The method according to any one of claims 17 to 22, wherein the cell is a cell into which DNA encoding a glycoprotein has been introduced.
[24] 糖蛋白質がアンチトロンビンである、請求項 17〜23のいずれ力、 1項に記載の方法。 [24] The method according to any one of [17] to [23], wherein the glycoprotein is antithrombin.
[25] 5〜200mmol/Lのシアル酸、 0. ；!〜 200mmol/Lのシアル酸重合物、；!〜 200 mmol/Lのシアル酸を含むオリゴ糖から選ばれる少なくとも 1種類の物質を添加した 培地中で、糖蛋白質組成物を生産する能力を有する細胞を培養することを特徴とす る、該細胞より生産される糖蛋白質組成物に結合する N—グリコリルノィラミン酸量を 抑制させる方法。 [25] 5 to 200 mmol / L sialic acid, 0.;! To 200 mmol / L sialic acid polymer;! To at least one substance selected from oligosaccharides containing 200 to 200 mmol / L sialic acid In the medium, cells having the ability to produce a glycoprotein composition are cultured, and the amount of N-glycolylneuraminic acid bound to the glycoprotein composition produced from the cells is suppressed. Method.
[26] シアル酸が N—ァセチルノイラミン酸である、請求項 25に記載の方法。  26. The method according to claim 25, wherein the sialic acid is N-acetylneuraminic acid.
[27] シアル酸、シアル酸重合物およびシアル酸を含むオリゴ糖から選ばれる少なくとも 1 種類の物質を培養開始時、対数増殖期、もしくは静止期に培地へ添加する、請求項 [27] The at least one substance selected from sialic acid, a sialic acid polymer and an oligosaccharide containing sialic acid is added to the medium at the start of culture, in the logarithmic growth phase, or in the stationary phase.
25または 26に記載の方法。 The method according to 25 or 26.
[28] 細胞を浸透圧 250〜400mOsm/kgで培養する、請求項 25〜27のいずれ力、 1項 に記載の方法。 [28] The method according to any one of [25] to [27], wherein the cells are cultured at an osmotic pressure of 250 to 400 mOsm / kg.
[29] 細胞が動物細胞である、請求項 25〜28のいずれか 1項に記載の方法。  [29] The method according to any one of claims 25 to 28, wherein the cells are animal cells.
[30] 動物細胞が、以下の（a)〜（l)からなる群から選ばれる細胞である、請求項 29に記載 の方法。  [30] The method according to claim 29, wherein the animal cell is a cell selected from the group consisting of the following (a) to (l).
(a)チャイニーズノ、ムスター卵巣組織由来 CHO細胞；  (a) Chinese CHO, derived from Muster ovarian tissue;
(b)ラットミエ口一マ糸田月包ネ朱 YB2/3HL. P2. Gi l . 16Ag. 20糸田月包；  (b) Rat Mye Kouichi Ma Itohtsuki Parcel Zhu YB2 / 3HL. P2. Gi l. 16Ag.
(c)マウスミエローマ細胞株 NS0細胞；  (c) mouse myeloma cell line NS0 cells;
(d)マウスミエローマ細胞株 SP2/0— Agl4細胞；  (d) mouse myeloma cell line SP2 / 0—Agl4 cells;
(e)シリアンノ、ムスター腎臓組織由来 BHK細胞；  (e) Syrianno, Muster kidney tissue-derived BHK cells;
(f)抗体を産生するハイプリドーマ細胞；  (f) a hyperidoma cell producing the antibody;
(g)ヒト白血病細胞株ナマルバ細胞；  (g) human leukemia cell line Namalva cells;
(h)ヒト白血病細胞株 NM— F9細胞；  (h) human leukemia cell line NM-F9 cells;
(i)ヒト胚性網膜細胞株 PER. C6細胞；  (i) human embryonic retinal cell line PER. C6 cells;
(j)胚性幹細胞；  (j) embryonic stem cells;
(k)受精卵細胞；  (k) a fertilized egg cell;
(1)ヒ卜 HEK293細胞。  (1) Herbal HEK293 cells.
[31] 細胞が、糖蛋白質をコードする DNAが導入された細胞である、請求項 25〜30のい ずれか 1項に記載の方法。 [31] The method according to any one of claims 25 to 30, wherein the cell is a cell into which DNA encoding a glycoprotein has been introduced. The method according to item 1.
[32] 糖蛋白質がアンチトロンビンである、請求項 25〜31のいずれか 1項に記載の方法。 [32] The method according to any one of claims 25 to 31, wherein the glycoprotein is antithrombin.
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