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WO2018159842A1 - Procédé de production de composition de darbépoétine, et procédé de culture pour cellules produisant de la darbépoétine - Google Patents

Procédé de production de composition de darbépoétine, et procédé de culture pour cellules produisant de la darbépoétine Download PDF

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WO2018159842A1
WO2018159842A1 PCT/JP2018/008156 JP2018008156W WO2018159842A1 WO 2018159842 A1 WO2018159842 A1 WO 2018159842A1 JP 2018008156 W JP2018008156 W JP 2018008156W WO 2018159842 A1 WO2018159842 A1 WO 2018159842A1
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darbepoetin
medium
cells
composition
culture
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PCT/JP2018/008156
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Japanese (ja)
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正義 塚原
耕一 山本
石原　尚
眞礼登 細野
泰志郎 中川
直史 奥村
智子 磯田
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協和発酵キリン株式会社
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Priority to KR1020197025072A priority Critical patent/KR102653666B1/ko
Priority to CN201880015112.0A priority patent/CN110462055A/zh
Publication of WO2018159842A1 publication Critical patent/WO2018159842A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/18—Ion-exchange chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/20—Partition-, reverse-phase or hydrophobic interaction chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/505—Erythropoietin [EPO]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

  • the present invention relates to a method for producing a darbepoetin composition, a method for culturing animal cells having the ability to produce darbepoetin, and the like.
  • glycoproteins are one of the genetically engineered proteins that are still difficult to produce even if genetic engineering technology advances. Many proteins exist as glycoproteins with added sugar chains, and it is known that the difference in the sugar chain structure greatly affects the function of the protein itself.
  • Biopharmaceuticals using genetic recombination technology are produced by introducing human protein genes into animal cells.
  • the sugar chain structure is not uniform due to sugar chain-degrading enzymes such as sialidase produced from the production cells, so that there may be cases where sufficient drug efficacy is not exhibited.
  • Erythropoietin a hematopoietic factor
  • EPO Erythropoietin
  • the blood half-life is decreased and hardly exhibits physiological activity.
  • darbepoetin has been found in which part of the amino acid sequence of EPO is modified for the purpose of further extending the half-life in blood of EPO and the number of sialic acids added is increased (Non-patent Document 2, Patent). Reference 1).
  • Darbepoetin has five N-linked sugar chains and one O-linked sugar chain, has a continuous erythrocyte-increasing action, and can reduce administration frequency compared to conventional EPO preparations. It becomes possible and the burden on the patient can be reduced.
  • the sugar chain control method includes, for example, 2-deoxy-2,3-dehydro, which is an inhibitor of sialidase, in order to inhibit elimination of sialic acid from glycoproteins by glycolytic enzymes secreted into the culture medium.
  • a method of culturing by adding a low molecular weight compound such as -N-acetylneuraminic acid (NeuAc2en) to the medium is known.
  • the medium conventionally used for culturing the animal cells is serum or serum-derived such as albumin, transferrin or insulin. Substances and animal-derived proteins have been added.
  • the addition of serum or serum-derived substances or animal-derived proteins to the culture medium may result in the risk of contamination of the cell culture and the product produced from it by hepatitis, viruses, bovine spongiform encephalopathy (BSE), etc. Therefore, development of a completely synthetic medium that does not contain serum or serum-derived substances and animal-derived proteins has been carried out.
  • the components of a typical fully synthetic medium include amino acids, organic or inorganic salts, vitamins, minerals, trace metals, sugars, lipids, nucleic acids, etc., but plant-derived protein hydrolysates such as soybeans It is known that by adding to a completely synthetic medium, the growth of animal cells and the production amount of the product obtained from the animal cells can be improved in the same manner as a serum-containing medium (Patent Documents 2 and 3). .
  • the present invention provides a method for producing a darbepoetin composition having a high sialic acid addition number by culturing darbepoetin-producing cells, and improving the total production amount of the darbepoetin composition secreted from the cells into the culture solution, and Another object of the present invention is to provide a method for producing a darbepoetin composition that improves the sialic acid content in one molecule of darbepoetin secreted from the cells into the culture medium.
  • the present inventors improve the total production amount of the darbepoetin composition secreted into the culture medium from the cells, Further, by inhibiting sialidase secreted into the culture solution, the sialic acid addition rate in one molecule of darbepoetin secreted into the culture solution from the cells is improved, and a darbepoetin composition having a high sialic acid addition number is markedly increased.
  • the present invention has been completed by finding that it can be produced.
  • the present invention provides the following (1) to (19).
  • (1) A method for producing a darbepoetin composition comprising the following steps 1 and 2.
  • Step 1 Step of culturing darbepoetin-producing cells in a medium to which a plant-derived protein hydrolyzate has been added to obtain a culture solution
  • Step 2 Step of recovering a darbepoetin composition from the culture solution obtained in Step 1
  • Step 1 Step of culturing darbepoetin-producing cells in a medium to which a plant-derived protein hydrolyzate has been added to obtain a culture solution
  • Step 2 Step of recovering a darbepoetin composition from the culture solution obtained in Step 1 (3 ) 67% or more of the darbepoetin composition containing 12-22 sialic acid per molecule of darbepoetin in the culture medium obtained in step 1 contains darbepoetin composition containing 18-22 sialic acid per darbepoetin molecule
  • the production method according to (1) or (2), which is a product is a product.
  • the total production amount of the darbepoetin composition in the culture solution obtained in step 1 is higher than that in culture in a plant-derived protein hydrolyzate-free medium, any of (1) to (6) 2.
  • the darbepoetin-producing cell is a transformed cell in which a vector containing a gene encoding darbepoetin is introduced into a host cell.
  • the host cell is an animal cell.
  • the animal cell is a Chinese hamster ovary (CHO) cell, a mouse melanoma (NS0) cell or a mouse myeloma (SP2 / 0) cell, or a cell derived from these cells. Production method.
  • the production method according to any one of (1) to (10), wherein the medium to which the plant-derived protein hydrolyzate is added is a completely synthetic medium.
  • the production method according to any one of (1) to (11), wherein the concentration of the plant-derived protein hydrolyzate added to the medium is 1 to 5 g / l.
  • the obtained darbepoetin composition is darbepoetin containing an average of 18 or more sialic acids per molecule.
  • a method for culturing darbepoetin-producing cells comprising culturing darbepoetin-producing cells in a medium to which a plant-derived protein hydrolyzate has been added and secreting the darbepoetin composition into the culture medium.
  • a method for culturing darbepoetin-producing cells comprising culturing darbepoetin-producing cells in a medium to which a plant-derived protein hydrolyzate has been added, and inhibiting sialic acid detachment from the darbepoetin molecules in the culture.
  • a method for inhibiting detachment of sialic acid from a darbepoetin molecule during storage of a culture solution comprising culturing darbepoetin-producing cells in a medium to which a plant-derived protein hydrolyzate has been added to obtain a culture solution .
  • culturing darbepoetin-producing cells in a medium to which a plant-derived protein hydrolyzate has been added improves the total production amount of the darbepoetin composition secreted from the cells into the culture medium.
  • the sialic acid content in one molecule of darbepoetin secreted from the cells into the culture medium can be improved.
  • FIG. 1 is a diagram showing the structure of darbepoetin.
  • FIG. 2 is a diagram showing the structures of N-linked and O-linked sugar chains of darbepoetin.
  • NeuAc represents N-acetylneuraminic acid, which is one of sialic acids.
  • FIG. 3 is a graph showing changes with time of the sialic acid isoform distribution of darbepoetin in a culture supernatant cultured in a soybean hydrolyzate-added medium and a soybean hydrolyzate removing solution.
  • Lanes 1 and 14 show darbepotin with 18-22 sialic acid additions.
  • Lane 2 stores soybean hydrolyzate removal liquid at 8 ° C.
  • Lane 3 stores soybean hydrolyzate removal liquid at 8 ° C. for 24 hours
  • Lane 4 stores soybean hydrolyzate removal liquid at 8 ° C. for 48 hours
  • Lane 5 shows the sialic acid isoform distribution when the soybean hydrolyzate removal solution is stored at 8 ° C. for 72 hours.
  • Lane 6 is a culture supernatant cultured in soybean hydrolyzate-containing medium at 8 ° C. for 0 hours
  • Lane 7 is a culture supernatant cultured in soybean hydrolyzate-containing medium at 8 ° C. for 24 hours
  • Lane 8 is soybean The culture supernatant cultured in the hydrolyzate-containing medium is stored at 8 ° C. for 48 hours.
  • Lane 9 shows the sialic acid isoform distribution when the culture supernatant cultured in the soy hydrolyzate-containing medium is stored at 8 ° C. for 72 hours. Each is shown.
  • the sialidase inhibitor NeuAc2en is added to the soybean hydrolyzate removal solution and stored at 8 ° C. for 0 hour
  • the sialidase inhibitor NeuAc2en is added to the soybean hydrolyzate removal solution for 24 hours at 8 ° C.
  • lane 12 added sialidase inhibitor NeuAc2en to soybean hydrolyzate removal solution and stored at 8 ° C. for 48 hours
  • lane 13 added sialidase inhibitor NeuAc2en to soybean hydrolyzate removal solution at 8 ° C.
  • FIG. 4 is a bar graph showing the results of Table 1 in which the ratio (%) of the area value of each sialic acid addition number darbepoetin composition to the total area value of the darbepoetin composition having 12 to 22 sialic acid additions was measured. Is. A vertical axis
  • shaft shows the ratio (%) of the area value of the darbepoetin composition of each sialic acid addition number.
  • the ratio of the area value of 22 sialic acid additions in Table 1 is the ratio of the area values of 22, 22-1S and 22-2S isoforms indicating the darbepoetin composition having 22 sialic acid additions in FIG. It is shown as the sum of (%).
  • the method for producing a darbepoetin composition of the present invention includes the following step 1 and step 2.
  • Step 1 Step of culturing darbepoetin-producing cells in a medium to which a plant-derived protein hydrolyzate has been added to obtain a culture solution
  • Step 2 Step of recovering a darbepoetin composition from the culture solution obtained in Step 1 Each process will be described.
  • Step 1 is a step of culturing cells having the ability to produce darbepoetin in a medium to which a plant-derived protein hydrolyzate is added, and secreting the darbepoetin composition into the medium to obtain a culture solution.
  • darbepoetin was substituted with Asn, Thr, Val, Asn and Thr in WO 95/05465 in the amino acid sequence of human erythropoietin at positions 30, 32, 87, 88 and 90, respectively.
  • [Asn 30 Thr 32 Val 87 Asn 88 Thr 90 ] is a human erythropoietin analog shown as EPO and has the structure shown in FIG.
  • N-linked sugar chains are bound to Asn residues at positions 24, 30, 38, 83 and 88 of darbepoetin, and O-linked sugar chains are bound to Ser residues at position 126.
  • the main structure of the type sugar chain and the O-linked sugar chain includes the structure shown in FIG.
  • the N-linked sugar chain that binds to the darbepoetin molecule contains up to 4 sialic acids, and the end of the O-linked sugar chain contains up to 2 sialic acids.
  • One darbepoetin molecule contains up to 22 sialic acids. Of sialic acid.
  • darbepoetin includes, for example, darbepoetin alfa or darbepoetin alfa (genetical recombination) or biosuccessors thereof.
  • novel erythropoiesis stimulating protein and NESP (trademark) or Aranesp (trademark) disclosed in International Publication No. 2003/020299, or their biosequential products are also included in the darbepoetin of the present invention.
  • the host cell of the darbepoetin-producing cell used in the present invention may be any animal cell belonging to any one of mammals, birds, reptiles, amphibians, fish and insects.
  • animal cells derived from primates such as humans or monkeys or animal cells derived from rodents such as mice, rats or hamsters are used.
  • Examples of cells belonging to mammals include myeloma cells or cells derived from myeloma cells, ovary cells, kidney cells, blood cell cells, uterine cell connective tissue cells, mammary cells, or embryonic retinoblasts.
  • myeloma cells or cells derived from myeloma cells and ovarian cells are preferable.
  • cells selected from myeloma cells or cells derived from myeloma cells and ovarian cells are preferable.
  • Chinese hamster ovary (CHO) cells, mouse melanoma (NS0) cells, or mouse myeloma (SP2 / 0) Cells or cells derived from these cells.
  • Examples of cells belonging to mammals include human cell lines HL-60 (ATCC CCL-240), HT-1080 (ATCC CCL-121), HeLa (ATCC CCL-2), 293 (ECACC 85120602), Namalwa ( ATCC CRL-1432), Namalwa KJM-1 (Cytotechnology, 1, 151 (1988)), NM-F9 (DSM ACC2605, International Publication No. 05/17130) or PER. C6 (ECACC No. 9602940, US Pat. No.
  • VERO ATCC CCL-1651
  • COS-7 ATCC CRL-1651
  • a monkey cell line C127I
  • a mouse cell line a monkey cell line
  • Sp2 / 0-Ag14 ATCC CRL-1581
  • NIH3T3 ATCCCRL-1658
  • NS0 ATCC CRL-1827
  • CHO-K1 (ATCC CCL-61) is a hamster cell lines, CHO / dhfr - (ATCC CRL -9096), CHO / DG44 [Proc. Natl. Acad. Sci. USA, 77, 4216 (1980)] or BHK21 (ATCC CRL-10) or MDCK (ATCC CCL-34) which is a canine cell.
  • Examples of cells belonging to birds include chicken cell line SL-29 (ATCC CRL-29), and examples of cells belonging to fish include zebrafish cell line ZF4 (ATCC CRL-2050). Examples thereof include Spodoptera frugiperda cell line Sf9 (ATCC CRL-1711). Examples of primary cultured cells include primary monkey kidney cells, primary rabbit kidney cells, primary chicken fetal cells, and primary quail fetal cells.
  • myeloma cells or cells derived from myeloma cells include Sp2 / 0-Ag14, NS0, Y3 Ag1.2.3. , YO or YB2 / 0.
  • the cells derived from the ovary cells or ovarian cells e.g., CHO-K1, CHO / dhfr - like or CHO / DG44.
  • kidney cells examples include 293, VERO, COS-7, BHK21, and MDCK.
  • blood cells examples include HL-60, Namalwa, Namalwa KJM-1, and NM-F9.
  • uterine cells include uterine cells. , HeLa, etc., as connective tissue cells, for example, HT-1080 or NIH3T3, etc., as mammary cells, for example, C1271I, etc., as embryonic retinoblasts, for example, PER. C6 etc. are mentioned, respectively.
  • Examples of the darbepoetin-producing cell include a transformed cell in which a vector containing a gene encoding darbepoetin is introduced into the above-described host cell, a cell that has been mutated to produce darbepoetin, or darbepoetin. Examples include hybridomas that are fusion cells of cells and myeloma cells. Furthermore, the darbepoetin-producing cells of the present invention include cells obtained by subjecting the above-described cells to a mutation treatment that increases the expression level of darbepoetin.
  • a recombinant vector containing a DNA involved in the production of darbepoetin and a promoter is introduced into the host cell used in the present invention described above. Obtained by.
  • DNA involved in the production of darbepoetin for example, DNA encoding darbepoetin, DNA encoding an enzyme or protein involved in biosynthesis of darbepoetin, and the like can be used.
  • Examples of the vector used for preparing the recombinant vector include pcDNAI, pcDM8 (manufactured by Funakoshi), pAGE107 [Japanese Unexamined Patent Publication No. 3-22979, Cytotechnology, 3, 133 (1990)], pAS3. -3 (Japanese Patent Laid-Open No. 2-227075), pcDM8 [Nature, 329, 840 (1987)], pcDNAI / Amp (manufactured by Invitrogen), pREP4 (manufactured by Invitrogen), pAGE103 [J. Biochem. , 101, 1307 (1987)], or pAGE210.
  • CMV cytomegalovirus
  • SV40 early promoter SV40 early promoter
  • retrovirus promoter examples include promoters, metallothionein promoters, heat shock promoters, SR ⁇ promoters, and the like.
  • an enhancer of IE gene of human CMV may be used together with a promoter.
  • any method can be used as long as it is a method for introducing DNA into the cell.
  • an electroporation method [Cytotechnology, 3, 133 (1990)].
  • Calcium phosphate method Japanese Patent Laid-Open No. 2-227075
  • lipofection method Japanese Patent Laid-Open No. 2-227075
  • any protein hydrolyzate can be used as long as it is a protein hydrolyzate derived from plants such as wheat, rice or soybean, but protein hydrolyzate derived from soybean is particularly preferable. .
  • a plant-derived protein hydrolyzate is a mixture containing, as a main component, short-chain peptides such as amino acids and dipeptides or tripeptides obtained by degrading proteins obtained from plants as raw materials, and trace metals. .
  • an acid decomposition method using an acid such as hydrochloric acid
  • an enzyme decomposition method using an enzyme such as a protease can be mentioned, and membrane treatment is performed as necessary.
  • the average molecular weight of the plant-derived protein hydrolyzate is preferably 800 Da or less, stepwise, more preferably 600 Da or less, still more preferably 450 Da or less, and particularly preferably 300 Da or less.
  • the molecular weight distribution of the plant-derived protein hydrolyzate is preferably ⁇ 500 Da, preferably 45-80%, 500-1000 Da, preferably 15-30%, 1000-2000 Da, preferably 5-15%, 2000-5000 Da. Is 2 to 10%.
  • ⁇ 2000 Da is more preferably 90 to 98%, and ⁇ 2000 Da is further preferably 95 to 98%.
  • protein hydrolysates derived from soybean include Amysoy, Hy-Soy, NZ-Soy, HyPep (hereinafter, Quest International), Soy Pettone (Gibco), Bac-Soyton (Difco), SE50MK ( DMV International Nutritions), Peptone Hy-Soy T, or Soy Hydrolysate UF (from Sigma-Aldrich), and other protein hydrolysates derived from wheat or rice include HyPep (Quest International).
  • the timing of adding the plant-derived protein hydrolyzate to the culture solution is not particularly limited at the start of culture or during the culture, but it is preferably added to the medium at the start of culture.
  • the plant-derived protein hydrolyzate may be added to the medium alone as a feed medium, or previously mixed with other medium components. It may be added to the medium as a prepared feed medium.
  • the culture period is not particularly limited, but the final main culture period is preferably 8 days or longer.
  • the main culture period is preferably 8 days to 1 month, more preferably 10 days to 21 days, and particularly preferably 10 days to 14 days.
  • the concentration of the plant-derived protein hydrolyzate added to the medium may be appropriately selected depending on the type of cells used for the culture, the timing of addition of the plant-derived protein hydrolyzate, and preferably 0.1 to 100 g. / L, more preferably 1 to 10 g / L, particularly preferably 1 to 5 g / L.
  • the medium used in the present invention may be any medium as long as it can be used for culturing darbepoetin-producing cells, but a completely synthetic medium containing no serum or serum-derived substance or animal-derived protein should be used. Is preferred.
  • basal medium used for normal animal cell culture is used as the basal medium.
  • basal media used for normal animal cell culture include, for example, RPMI 1640 medium [The Journal of the American Medical Association, 199, 519 (1967)], Eagle's MEM medium [Science, 122, 501 (1952)], Dulbecco's modified MEM (DMEM) medium [Virology, 8, 396 (1959)], 199 medium [Proceeding of the Society for the Biologic Medicine, 73, 1 (1950)], F12 medium (manufactured by LTI) [Proc. Natl. Acad. Sci.
  • Iskov modified Dulbecco medium IMDM medium
  • J. Experimental Medicine 147, 923 (1978)
  • EX-CELL325PF medium manufactured by JRH
  • modified medium or mixed medium thereof preferably RPMI1640 medium, DMEM medium, F12 medium, IMDM medium or EX- CELL 325PF medium or the like is used.
  • a nutrient factor or a physiologically active substance necessary for the growth of animal cells is added to the basal medium as necessary.
  • These additives are preferably contained in the medium in advance before culturing.
  • nutritional factors examples include sugars such as glucose, amino acids, vitamins, and the like.
  • amino acids examples include L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cystine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L -Lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine and the like are used, and one kind or a combination of two or more kinds is used.
  • vitamins include d-biotin, D-pantothenic acid, choline, folic acid, myo-inositol, niacinamide, pyridoxal, riboflavin, thiamine, cyanocobalamin, and DL- ⁇ -tocopherol. Used in combination.
  • substances that are added in place of animal-derived substances in a completely synthetic medium that does not contain animal-derived substances do not include, for example, physiologically active substances, hydrolysates, or animal-derived substances produced by genetic recombination methods. Examples thereof include lipids, minerals, trace metals, and nucleic acids. Examples of the physiologically active substance produced by the gene recombination method include gene recombinant insulin, gene transferrin, gene recombinant albumin, gene recombinant growth factor, and the like.
  • lipids that do not contain animal-derived substances include cholesterol, linoleic acid, and linolenic acid.
  • ADPF medium (Animated protein free medium; HyClone), CD-Hybridoma medium (Invitrogen), CD-CHO medium (Invitrogen), IS CD-CHO medium, or KINSM- 10 medium (Irvine Scientific).
  • a medium containing a high concentration of amino acids and vitamins such as a medium in which RPMI1640 medium, DMEM medium and F12 medium are mixed at a ratio of 1: 1: 1, DMEM medium and F12 A medium in which the medium is mixed at a ratio of 1: 1, a hybridoma SFM medium (manufactured by Invitrogen) and the like are preferably used.
  • the total production amount of the darbepoetin composition in the culture solution obtained in step 1 is improved when the culture is completed in a medium containing the above-mentioned plant-derived protein hydrolyzate. It means that the concentration of the darbepoetin composition in the unit culture volume is improved as compared with the case of culturing in the above plant-derived hydrolyzate-free medium.
  • the concentration of the darbepoetin composition in the unit culture volume can be determined using any known protein concentration measurement method such as ELISA or HPLC, but for example, Biacore (GE Healthcare) can be used.
  • the protein concentration measuring method used is mentioned.
  • sialic acid is preferably 18-22, more preferably 19-22, particularly preferably 20-22, most preferably 22 per molecule of darbepoetin. It has an added structure.
  • the darbepoetin composition in the culture solution obtained in step 1 preferably 67% or more of the darbepoetin composition containing 12 to 22 sialic acids per molecule of darbepoetin comprises 18 to 22 siaals per darbepoetin molecule. It is a darbepoetin composition containing an acid. Preferably, 55% or more of the darbepoetin composition containing 12-22 sialic acid per molecule of darbepoetin is a darbepoetin composition containing 19-22 sialic acid per molecule of darbepoetin.
  • the darbepoetin composition containing 12 to 22 sialic acid per molecule of darbepoetin is a darbepoetin composition containing 20 to 22 sialic acid per molecule of darbepoetin.
  • 18% or more of the darbepoetin composition containing 12 to 22 sialic acids per molecule of darbepoetin is a darbepoetin composition containing 22 sialic acids per molecule of darbepoetin.
  • the darbepoetin composition in the culture solution obtained in step 1 include, for example, preferably a darbepoetin composition containing 12 to 22 sialic acids per molecule of darbepoetin 10 to 14 days after the start of culture. Mention may be made of darbepoetin compositions in which more than 67% contain 18-22 sialic acids per molecule of darbepoetin. Further, specifically, for example, preferably 55% or more of the darbepoetin composition containing 12 to 22 sialic acids per molecule of darbepoetin on the 10th to 14th day after the start of the culture is 19 to 22 per darbepoetin molecule.
  • darbepoetin composition containing sialic acid preferably, at least 10% of darbepoetin composition containing 12-22 sialic acid per molecule of darbepoetin on the 10th to 14th day after the start of culturing is 20-22 per darbepoetin molecule.
  • a darbepoetin composition containing sialic acid preferably 18% or more of the darbepoetin composition containing 12 to 22 sialic acids per molecule of darbepoetin on the 10th to 14th day after the start of culturing is 22 siales per darbepoetin molecule.
  • a darbepoetin composition containing an acid may be mentioned.
  • the number of sialic acids in the darbepoetin composition can be determined by measuring the sialic acid isoform distribution of darbepoetin by an isoelectric focusing method using a capillary electrophoresis apparatus (manufactured by Beckman Coulter) or the like.
  • Step 2 is a step of recovering the darbepoetin composition from the culture solution obtained in Step 1.
  • the darbepoetin composition secreted into the culture medium by the culture in step 1 can be purified according to a known method, and the production method of the present invention may further include a purification step of purifying the darbepoetin composition.
  • a culture supernatant is obtained from the culture solution obtained in step 1 by a technique such as centrifugation, and from the culture supernatant, a normal recombinant protein isolation and purification method, that is, a solvent extraction method, ammonium sulfate, etc.
  • resin such as DIAION HPA-75 (Mitsubishi Chemical), S-Sepharose FF (Pharmacia) Cation exchange chromatography using resin such as butyl sepharose, or hydrophobic chromatography using resin such as phenyl sepharose,
  • the darbepoetin composition is preferably darbepoetin containing an average of 18 or more sialic acids per molecule, more preferably an average of 18.5 or more per molecule.
  • the darbepoetin composition is preferably, for example, preferably an average of 18 or more per molecule, more preferably an average of 18.5 or more per molecule, obtained from a culture solution 10 to 14 days after the start of culture. Examples include darbepoetin containing an acid.
  • the number of sialic acids in the darbepoetin composition is determined by measuring the sialic acid isoform distribution of darbepoetin using isoelectric focusing using a capillary electrophoresis apparatus (manufactured by Beckman Coulter) or the like. Can do.
  • the present invention relates to a method for culturing darbepoetin-producing cells, comprising culturing darbepoetin-producing cells in a medium to which a plant-derived protein hydrolyzate has been added and secreting the darbepoetin composition into the culture solution.
  • the culture of darbepoetin-producing cells in the culture method is the same as in step 1 described above.
  • the present invention also relates to a method for producing a darbepoetin-producing cell comprising culturing darbepoetin-producing cells in a medium to which a plant-derived protein hydrolyzate has been added, and inhibiting detachment of sialic acid from darbepoetin molecules in the culture.
  • the present invention relates to a culture method.
  • the culture of darbepoetin-producing cells in the culture method is the same as in step 1 described above.
  • the present invention relates to detachment of sialic acid from a darbepoetin molecule during storage of a culture solution, including culturing darbepoetin-producing cells in a medium supplemented with a plant-derived protein hydrolyzate to obtain a culture solution. It relates to a method of inhibiting. According to the method of the present invention, the action of the plant-derived protein hydrolyzate contained in the culture solution inhibits the elimination of sialic acid from the darbepoetin molecule by sialidase until purification is started after completion of the culture. The rate of sialic acid addition to darbepoetin molecules during storage of the culture medium can be improved.
  • save conditions of a culture solution are not specifically limited, Preferably it can preserve
  • Example 1 Comparison of sialic acid addition rate of darbepoetin composition produced in soybean hydrolyzate-containing medium and non-containing medium Using CHO cells producing darbepoetin, fed batch culture was performed in a 1 L Erlenmeyer flask. The sialic acid addition rate of darbepoetin was compared between the hydrolyzate-containing medium and the non-containing medium.
  • the darbepoetin-producing CHO cells were expanded from a 125 mL Erlenmeyer flask to a 500 mL Erlenmeyer flask using EX-CELL325PF medium (Sigma-Aldrich), and further expanded in a 1 L Erlenmeyer flask (Corning). Thus, a seed culture solution necessary for the main culture was obtained.
  • Expansion culture was carried out at 37 ° C. for 3 or 4 days by seeding the cells so that the amount of medium was about 10-30% of each flask volume to 2 ⁇ 10 5 cells / mL.
  • a medium based on KINSM-10 medium Irvine Scientific
  • 3 g / L of soybean hydrolyzate Soy Hydrateate UF Sigma-Aldrich; molecular weight distribution: ⁇ 500 Da is 66%, 500 to 1000 Da is 22 %, 1000-2000 Da 9%, 2000-5000 Da 3%) with or without (Soy) or added (without Soy)
  • the seed culture is centrifuged in a 1 L Erlenmeyer flask filled with 200 mL of production medium (1000 rpm, 25 ° C., 5 minutes), and the cells from which the supernatant was removed were seeded at 2.0 ⁇ 10 5 cells / mL.
  • the cell density immediately after sowing was 2.4 ⁇ 10 5 cells / mL in the soybean hydrolyzate-
  • the culture solution was collected, and the total production amount of the darbepoetin composition in the culture solution was measured using Biacore (GE Healthcare). Furthermore, about 600 ⁇ g of the darbepoetin composition in the collected sample was separated and obtained from these collected samples using a separator [AKTA explorer (manufactured by GE Healthcare)] having an anti-darbepoetin monoclonal antibody column.
  • AKTA explorer manufactured by GE Healthcare
  • the sialic acid isoform distribution of the obtained darbepoetin composition was measured using a capillary electrophoresis apparatus (manufactured by Beckman Coulter).
  • the Cartridge temperature was set to 25 ° C.
  • the voltage value was set to 6 kV
  • 10 mmol / L Tricine 10 mmol / L NaCl
  • 10 mmol / L sodium acetate 7 mmol / L UREA
  • 2.5 mmol / L Putrescine, pH 4 was performed using a buffer consisting of .7.
  • Table 1 and FIG. 4 show the results of measuring the ratio (%) of the area value of the darbepoetin composition having each sialic acid addition number to the total area value of the darbepoetin composition having 12 to 22 sialic acid additions.
  • the ratio of darbepoetin having 18 to 22 sialic acid additions was higher than that in the soybean hydrolyzate-free medium (no Soy). Increased. On the 14th day of culture, the ratio was 12.3% for darbepoetin with 18 sialic acid additions, 11.2% for darbepoetin with 19 sialic acid additions, and 12 for darbepoetin with 20 sialic acid additions.
  • darbepoetin with 21 sialic acid additions is 13.6%
  • darbepoetin with 22 sialic acid additions is 18.6%
  • the ratio of darbepoetin with 22 sialic acid additions is particularly remarkable Increased to.
  • the average sialic acid addition number per molecule was 18.7.
  • the total production amount of the darbepoetin composition having 12 to 22 sialic acid additions was about 1.7 to 2.2 times higher in the soybean hydrolyzate-containing medium than in the soybean hydrolyzate-free medium.
  • soybean hydrolyzate is effective as a medium composition for producing a darbepoetin composition having a large number of sialic acid additions.
  • Example 2 Inhibition of sialic acid elimination from darbepoetin molecules by soybean hydrolyzate in the culture medium
  • a culture supernatant cultured in a soybean hydrolyzate-containing medium prepared in the same manner as in Example 1 was tested at 0 ° C. The stability test was conducted after storage for ⁇ 72 hours. Further, the culture supernatant cultured in a soybean hydrolyzate-containing medium prepared in the same manner as in Example 1 was concentrated using a concentrated membrane (Amicon Ultra-4 30000MWCO, manufactured by Millipore) and then replaced with MilliQ water. A hydrolyzate removing solution was prepared and stored at 8 ° C. for 0 to 72 hours in the same manner.
  • a concentrated membrane Amicon Ultra-4 30000MWCO, manufactured by Millipore
  • soybean hydrolyzate in the medium inhibits sialic acid elimination from sialicase-added darbepoetin molecules.

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Abstract

La présente invention a pour objet de fournir un procédé de culture de cellules produisant de la darbépoétine et de produire ainsi une composition de darbépoétine, le procédé augmentant la quantité totale de la composition de darbépoétine produite et augmentant la teneur en acide sialique de chaque molécule de darbépoétine excrétée dans une solution de culture par les cellules. La présente invention concerne un procédé de production d'une composition de darbépoétine. Le procédé de production comprend : une première étape de culture de cellules produisant de la darbépoétine dans un milieu de culture auquel un hydrolysat de protéine d'origine végétale a été ajouté et l'obtention d'une solution de culture ; et une seconde étape de collecte de la composition de darbépoétine à partir de la solution de culture obtenue à l'étape 1.
PCT/JP2018/008156 2017-03-03 2018-03-02 Procédé de production de composition de darbépoétine, et procédé de culture pour cellules produisant de la darbépoétine WO2018159842A1 (fr)

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