WO2021090888A1 - タンパク質の製造方法 - Google Patents

タンパク質の製造方法 Download PDF

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Publication number
WO2021090888A1
WO2021090888A1 PCT/JP2020/041377 JP2020041377W WO2021090888A1 WO 2021090888 A1 WO2021090888 A1 WO 2021090888A1 JP 2020041377 W JP2020041377 W JP 2020041377W WO 2021090888 A1 WO2021090888 A1 WO 2021090888A1
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Prior art keywords
medium
concentration
culture
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phosphoric acid
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English (en)
French (fr)
Japanese (ja)
Inventor
靖道 古関
貴裕 片山
裕介 大矢
文 緒方
聖 平川
拓哉 樋口
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Ajinomoto Co Inc
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Ajinomoto Co Inc
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Priority to EP20885757.3A priority Critical patent/EP4056678A4/en
Priority to KR1020227017195A priority patent/KR20220097421A/ko
Priority to CN202080076873.4A priority patent/CN114667351A/zh
Priority to JP2021555106A priority patent/JP7513034B2/ja
Publication of WO2021090888A1 publication Critical patent/WO2021090888A1/ja
Priority to US17/730,750 priority patent/US12584156B2/en
Anticipated expiration legal-status Critical
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    • 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
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0018—Culture media for cell or tissue culture
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/241—Tumor Necrosis Factors
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12N5/0602—Vertebrate cells
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    • C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/12—Light metals, i.e. alkali, alkaline earth, Be, Al, Mg
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/32—Amino acids
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/42—Organic phosphate, e.g. beta glycerophosphate
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    • C12N2500/00—Specific components of cell culture medium
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    • C12N2500/46—Amines, e.g. putrescine
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
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    • C12N2510/00—Genetically modified cells
    • C12N2510/02—Cells for production

Definitions

  • the present invention relates to a method for culturing animal cells.
  • One aspect of the present invention relates to a method for producing a target substance such as a protein by an animal cell.
  • Animal cells are used as an expression host for recombinant proteins.
  • Patent Document 1 As a method for producing a recombinant protein as an expression host of a Chinese hamster ovary-derived cell line (CHO), a fed-batch medium containing phosphoric acid is flowed so that the phosphoric acid concentration in the medium is 1.5 to 3.5 mM. A method of adding has been reported (Patent Document 1).
  • Patent Document 2 a method of culturing cells using a medium containing a high concentration of choline
  • Patent Document 3 a method of culturing cells using a medium containing a high concentration of serine
  • Patent Document 4 A method of culturing cells using a medium containing an amine (Patent Document 4) is known.
  • An object of the present invention is to provide a novel technique for improving the culture performance of animal cells (for example, proliferation of animal cells and production of target substance by animal cells).
  • the present inventors increase the concentration of specific components such as phosphate and potassium in the medium when culturing animal cells to increase the culture results of animal cells (for example, proliferation of animal cells and target substances by animal cells).
  • the present invention has been completed by finding that the production) is improved.
  • the present invention can be exemplified as follows.
  • the method the culture is carried out under the conditions (A) and / or (B) below.
  • the method, wherein the target substance is a protein.
  • a method of culturing animal cells Including culturing animal cells in medium The method: the culture is carried out under the conditions (A) and / or (B) below.
  • the method of the present invention is a method for culturing animal cells, which comprises culturing the animal cells in a medium, and the culture enhances the phosphoric acid concentration and / or the potassium concentration in the medium. It is a method that is carried out under the above conditions.
  • the target substance may be produced by culturing animal cells. That is, when an animal cell has a target substance-producing ability, the target substance can be produced by culturing the cell. That is, one aspect of the method of the present invention is a method for producing a target substance, which comprises culturing an animal cell capable of producing the target substance in a medium and recovering the target substance, and the culture is the medium. The method may be carried out under conditions in which the concentration of phosphoric acid and / or the concentration of potassium in the medium is increased.
  • the target substance is not particularly limited as long as it can be produced by animal cells.
  • examples of the target substance include proteins.
  • a protein produced as a target substance is also referred to as a "target protein”.
  • Animal cells are not particularly limited. The animal cell can be appropriately selected depending on various conditions such as the use of the animal cell. For example, when an animal cell is used for producing a target protein, the animal cell is not particularly limited as long as it can express the target protein.
  • Animal cells are also referred to as "hosts," "expression hosts,” or "host cells.” Animals include mammals, birds and amphibians. Animals include mammals in particular. Mammals include rodents and primates. Rodents include hamsters, mice, rats and guinea pigs. Examples of hamsters include Chinese hamsters. Primates include humans, monkeys and chimpanzees. Examples of monkeys include African green monkeys. Examples of birds include chickens. Amphibians include Xenopus laevis.
  • tissue or cell from which the animal cell is derived is not particularly limited.
  • Tissues or cells from which animal cells are derived include ovaries, kidneys, adrenal glands, tongue epithelium, olfactory epithelium, pineal gland, thyroid gland, and melanocytes.
  • Examples of Chinese hamster cells include Chinese hamster ovary-derived cell lines (CHO). Specific examples of CHO include CHO-DG44 and CHO-K1. Examples of human cells include human fetal kidney cell-derived cell lines (HEK). Specific examples of HEK include HEK293 and HEK293T. Examples of African green monkey cells include African green monkey renal cell-derived cell lines (COS). Specific examples of COS include COS-1.
  • Specific examples of Xenopus cells include Xenopus oocytes.
  • Animal cell capable of producing a target substance means an animal cell capable of producing a target substance.
  • animal cells capable of producing a target substance means an ability to produce a target substance (for example, express a target protein) and accumulate it in a culture to a extent that it can be recovered when cultured in a medium. It may mean an animal cell having.
  • "Accumulation in culture” may specifically mean accumulation in medium, cell surface, intracellular, or a combination thereof. The case where the target substance accumulates extracellularly (for example, in the medium or on the cell surface) is also referred to as "secretion" or "secretory production” of the target substance.
  • the animal cell may have a secretory production ability (the ability to secrete and produce the target substance) of the target substance.
  • the accumulated amount of the target substance may be, for example, 10 ⁇ g / L or more, 1 mg / L or more, 100 mg / L or more, or 1 g / L or more as the accumulated amount in the culture.
  • the animal cell may have the ability to produce one kind of target substance, or may have the ability to produce two or more kinds of target substances.
  • the animal cell may be one that originally has the ability to produce the target substance, or may be one that has been modified to have the ability to produce the target substance.
  • the animal cells may be modified so as to enhance the inherent ability to produce the target substance.
  • the animal cell having the target substance-producing ability can be obtained, for example, by imparting the target substance-producing ability to the animal cell as described above, or by enhancing the target substance-producing ability of the animal cell as described above. ..
  • the target protein-producing ability can be imparted or enhanced by introducing a gene encoding the target protein.
  • the gene encoding the target protein is also referred to as the "target protein gene".
  • the target protein is not particularly limited as long as it can be expressed using animal cells as a host.
  • the protein may be a host-derived protein or a heterologous protein.
  • the "heterologous protein” refers to a protein that is exogenous to a host that produces the protein (ie, an animal cell capable of producing the protein of interest).
  • the target protein may be, for example, a naturally occurring protein, a modified protein thereof, or an artificially designed amino acid sequence protein.
  • the target protein may be, for example, a protein derived from a microorganism, a protein derived from a plant, a protein derived from an animal, or a protein derived from a virus.
  • the protein of interest may be, in particular, a protein of human origin.
  • the target protein may be a monomeric protein or a multimeric protein.
  • the target protein may be a secretory protein or a non-secretory protein.
  • the "protein” also includes what is called a peptide, such as an oligopeptide or a polypeptide.
  • target protein examples include enzymes, bioactive proteins, receptor proteins, antigen proteins, and other proteins.
  • Enzymes include cellulase, transglutaminase, protein glutaminase, isomaltodextranase, protease, endopeptidase, exopeptidase, aminopeptidase, carboxypeptidase, collagenase, and chitinase.
  • bioactive proteins include growth factors (growth factors), hormones, cytokines, and antibody-related molecules.
  • Growth factors include epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), and transforming growth factor (TGF). ), Nerve growth factor (NGF), Brain-derived neurotrophic factor (BDNF), Vascular endothelial growth factor (VEGF), Granulocyte- colony stimulating factor (G-CSF), granulocyte-macrophage-colony stimulating factor (GM-CSF), platelet-derived growth factor (PDGF), erythropoietin (EPO), Thrombopoietin (TPO), acidic fibroblast growth factor (aFGF or FGF1), basic fibroblast growth factor (bFGF or FGF2), keratinocyte growth factor ; KGF-1 or FGF7, KGF-2 or FGF10), hepatocellular growth factor (HGF), stem cell factor (SCF), activin (Activin). Examples of activin include activin A, C, and E.
  • hormones include insulin, glucagon, somatostatin, human growth hormone (hGH), parathyroid hormone (PTH), calcitonin, and exenatide.
  • Cytokines include interleukin, interferon, and tumor necrosis factor (TNF).
  • the bioactive protein may be the whole protein or a part thereof.
  • a portion having a physiological activity can be mentioned.
  • Specific examples of the portion having physiological activity include the physiologically active peptide Teriparatide consisting of 34 amino acid residues at the N-terminal of a mature parathyroid hormone (PTH).
  • Antibody-related molecule may mean a protein comprising a molecular species consisting of a single domain selected from the domains constituting a complete antibody or a combination of two or more domains. Domains that make up a complete antibody include the heavy chain domains VH, CH1, CH2, and CH3, and the light chain domains VL and CL.
  • the antibody-related molecule may be a monomeric protein or a multimeric protein as long as it contains the above-mentioned molecular species. When the antibody-related molecule is a multimeric protein, it may be a homomultimer composed of a single type of subunit, or a heteromultimer composed of two or more types of subunits. May be good.
  • antibody-related molecules include complete antibodies, Fab, F (ab'), F (ab') 2 , Fc, dimers consisting of heavy chain (H chain) and light chain (L chain), and Fc. Fusion protein, heavy chain (H chain), light chain (L chain), single chain Fv (scFv), sc (Fv) 2 , disulfide bond Fv (sdFv), diabody, VHH fragment (nanobody®) Be done. More specific examples of antibody-related molecules include Trastuzumab, Adalimumab, and Nivolumab.
  • the receptor protein examples include a physiologically active protein and a receptor protein for other physiologically active substances.
  • Other bioactive substances include neurotransmitters such as dopamine.
  • the receptor protein may be an orphan receptor whose corresponding ligand is unknown.
  • the antigen protein is not particularly limited as long as it can elicit an immune response.
  • the antigen protein can be appropriately selected, for example, according to the target of the assumed immune response.
  • the antigen protein can be used, for example, as a vaccine.
  • LFABP Liver-type fatty acid-binding protein
  • fluorescent protein include Green Fluorescent Protein (GFP).
  • immunoglobulin binding protein include Protein A, Protein G, and Protein L.
  • albumin include human serum albumin.
  • fibroin-like protein include those disclosed in WO2017 / 090665 and WO2017 / 171001.
  • Laminin is a protein having a heterotrimeric structure consisting of ⁇ chain, ⁇ chain, and ⁇ chain.
  • laminin include mammalian laminin.
  • the laminin subunit chains ie, ⁇ chain, ⁇ chain, and ⁇ chain
  • the laminin subunit chains include 5 types of ⁇ chain ( ⁇ 1 to ⁇ 5), 3 types of ⁇ chain ( ⁇ 1 to ⁇ 3), and 3 types of ⁇ chain ( ⁇ 1). ⁇ ⁇ 3) can be mentioned.
  • Laminin constitutes various isoforms by combining these subunit chains.
  • laminin specifically, for example, laminin 111, laminin 121, laminin 211, laminin 213, laminin 221, laminin 311, laminin 321, laminin 332, laminin 411, laminin 421, laminin 423, laminin 511, laminin 521, laminin. 523 can be mentioned.
  • a partial sequence of laminin includes laminin E8, which is an E8 fragment of laminin.
  • laminin E8 has a heterotrimeric structure consisting of an ⁇ chain E8 fragment ( ⁇ chain E8), a ⁇ chain E8 fragment ( ⁇ chain E8), and a ⁇ chain E8 fragment ( ⁇ chain E8). It is a protein that has.
  • the subunit chains of laminin E8 are collectively referred to as “E8 subunit chains”.
  • Examples of the E8 subunit chain include the E8 fragment of the laminin subunit chain exemplified above.
  • Laminin E8 constitutes various isoforms by combining these E8 subunit chains.
  • Specific examples of the laminin E8 include laminin 111E8, laminin 121E8, laminin 211E8, laminin 221E8, laminin 332E8, laminin 421E8, laminin 411E8, laminin 511E8, and laminin 521E8.
  • the target protein may be, for example, a protein having a known or natural amino acid sequence of the above-mentioned protein. Further, the target protein may be, for example, a variant of a protein having a known or natural amino acid sequence of the above-mentioned protein. Variants include proteins having an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added at one or several positions in a known or natural amino acid sequence. Specifically, "1 or several" means, for example, 1 to 50, 1 to 40, 1 to 30, preferably 1 to 20, more preferably 1 to 10, and even more preferably 1. It may mean up to 5, particularly preferably 1 to 3.
  • Variants include, for example, 50% or more, 65% or more, 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 97% or more, particularly with respect to the entire known or natural amino acid sequence.
  • a protein having an amino acid sequence having 99% or more identity is also mentioned.
  • the protein specified by the species of origin is not limited to the protein itself found in the species, but includes proteins having the amino acid sequence of the protein found in the species and variants thereof.
  • Variants may or may not be found in the species. That is, for example, "human-derived protein” is not limited to the protein itself found in humans, but includes proteins having an amino acid sequence of proteins found in humans and variants thereof.
  • the target protein gene is not particularly limited as long as it encodes the target protein as described above.
  • the target protein gene may be, for example, a gene having a known or natural base sequence of a gene encoding a protein as described above. Further, the target protein gene may be, for example, a variant of a gene having a known or natural base sequence of a gene encoding a protein as described above.
  • the protein gene of interest may be modified, for example, to encode a protein having a variant sequence as exemplified above.
  • the target protein gene may be one in which any codon is replaced with an equivalent codon.
  • the target protein gene may be modified to have an optimum codon, for example, depending on the codon usage frequency of the host cell.
  • the term "gene” is not limited to DNA as long as it encodes the corresponding expression product, and may include any polynucleotide. That is, the "target protein gene” may mean any polynucleotide encoding the target protein.
  • the target protein gene may be DNA, RNA, or a combination thereof.
  • the target protein gene may be single-stranded or double-stranded.
  • the target protein gene may be single-stranded DNA or single-stranded RNA.
  • the target protein gene may be double-stranded DNA, double-stranded RNA, or a hybrid strand consisting of a DNA strand and an RNA strand.
  • the protein gene of interest may contain both DNA and RNA residues in a single polynucleotide strand.
  • the protein gene of interest may or may not contain an intron.
  • the mode of the target protein gene can be appropriately selected according to various conditions such as the means for expressing the target protein.
  • the expression “having a (amino acid or base) sequence” means “including the (amino acid or base) sequence” and includes the case of “consisting of the (amino acid or base) sequence”.
  • the target protein is expressed from the target protein gene. That is, an animal cell capable of producing a target protein has a target protein gene. Specifically, an animal cell having a target protein-producing ability has a target protein gene capable of expressing it. It is sufficient that the animal cell having the target protein-producing ability has the target protein gene until the target protein is expressed to a desired degree. That is, the animal cell capable of producing the target protein may or may not have the target protein gene after the expression of the target protein.
  • "expression of target protein gene” and "expression of target protein” may be used synonymously.
  • the target protein gene can be obtained by cloning from an organism having the target protein gene. Nucleic acids such as genomic DNA and cDNA containing the same gene can be used for cloning. The target protein gene can also be obtained by chemical synthesis (Gene, 60 (1), 115-127 (1987)).
  • the acquired target protein gene can be used as it is or after being appropriately modified. That is, the variant can be obtained by modifying the target protein gene.
  • the gene can be modified by a known method.
  • a site-specific mutation method can be used to introduce a desired mutation into a target site of DNA.
  • a site-specific mutation method a method using PCR (Higuchi, R., 61, in PCR technology, Erlich, H. A. Eds., Stockton press (1989); Carter, P., Meth. In Enzymol., 154, 382 (1987)) and methods using phage (Kramer, W. and Frits, H. J., Meth. In Enzymol., 154, 350 (1987); Kunkel, T. A. et al., Meth . In Enzymol., 154, 367 (1987)).
  • the variant of the target protein gene may be directly obtained by chemical synthesis.
  • the form of introducing the target protein gene into the host cell is not particularly limited.
  • the target protein gene may be retained in the host cell so that it can be expressed. Specifically, for example, when a target protein gene is introduced in a form that requires transcription of DNA or the like, the target protein gene is retained in a host cell so as to be expressible under the control of a promoter functioning in the host cell. Just do it.
  • the protein gene of interest may be extrachromosomally present or introduced onto the chromosome. When introducing two or more genes, each gene need only be retained in the host cell so that it can be expressed.
  • the promoter for expressing the target protein gene is not particularly limited as long as it functions in the host cell.
  • "Promoter functioning in a host cell” means a promoter having promoter activity in a host cell.
  • the promoter may be a host cell-derived promoter or a heterologous promoter.
  • the promoter may be a promoter unique to the target protein gene or a promoter of another gene.
  • the promoter may be a stronger promoter than the unique promoter of the target protein gene.
  • Promoters that function in animal cells include the SV40 promoter, EF1a promoter, RSV promoter, CMV promoter, and SRalpha promoter. Further, as the promoter, a highly active form of a conventional promoter may be obtained and used by using various reporter genes. Methods for assessing promoter strength and examples of potent promoters are described in Goldstein et al.'S paper (Prokaryotic promoters in biotechnology. Biotechnol. Annu. Rev., 1, 105-128
  • the target protein gene can be introduced into a host cell using, for example, a vector containing the same gene.
  • a vector containing a target protein gene is also referred to as an "expression vector of the target protein gene".
  • the expression vector of the target protein gene can be constructed, for example, by ligating a DNA fragment containing the target protein gene with the vector.
  • the gene By introducing the expression vector of the target protein gene into the host cell, the gene can be introduced into the host cell.
  • the vector may comprise a marker such as a drug resistance gene.
  • the vector may include an expression regulatory sequence such as a promoter for expressing the inserted gene.
  • the vector can be appropriately selected according to various conditions such as the type of host cell and the form of introduction of the target protein gene.
  • Examples of vectors that can be used for gene transfer into animal cells include plasmid vectors and viral vectors.
  • the viral vector include a retrovirus vector and an adenovirus vector.
  • Examples of the plasmid vector include a pcDNA series vector (pcDNA3.1 and the like; Thermo Fisher Scientific), a pBApo-CMV series vector (Takara Bio), and pCI-neo (Promega).
  • the vector can be integrated into the chromosome of the host cell, can autonomously replicate outside the chromosome of the host cell, or can be temporarily retained outside the chromosome of the host cell.
  • a vector having a viral origin of replication can replicate autonomously outside the chromosomes of animal cells.
  • the pcDNA series vector has an SV40 origin of replication and can autonomously replicate extrachromosomally in host cells expressing the SV40 large T antigen (COS-1, HEK293T, etc.).
  • the target protein gene can also be introduced into a host cell by, for example, introducing a nucleic acid fragment containing the gene into the host cell.
  • nucleic acid fragments include linear DNA and linear RNA.
  • linear RNA include mRNA and cRNA.
  • the method for introducing nucleic acids such as vectors and nucleic acid fragments into host cells can be appropriately selected according to various conditions such as the type of host cells.
  • Examples of the method for introducing a nucleic acid such as a vector or a nucleic acid fragment into an animal cell include a DEAE dextran method, a calcium phosphate method, a lipofection method, an electroporation method, and a microinjection method.
  • the vector is a viral vector
  • the vector can be introduced into the host cell by infecting the host cell with the vector (virus).
  • a cell that originally has the target protein gene may be modified and used so that the expression of the target protein gene is increased.
  • Increased gene expression means that the per-cell expression level of the gene is increased as compared to unmodified cells.
  • unmodified cell as used herein means a control cell that has not been modified to increase the expression of the target gene. Examples of unmodified cells include wild-type cells and cells from which they are modified.
  • Methods for increasing the expression of the target protein gene include increasing the number of copies of the target protein gene and improving the transcription efficiency and translation efficiency of the target protein gene. An increase in the number of copies of the target protein gene can be achieved by introducing the target protein gene into a host cell. The introduction of the target protein gene can be carried out as described above.
  • the target protein gene to be introduced may be derived from a host cell or a heterologous gene. Improvements in transcription efficiency and translation efficiency of the target protein gene can be achieved by modifying the expression regulatory sequences of genes such as promoters. For example, improvement in transcription efficiency of a target protein gene can be achieved by replacing the promoter of the target protein gene with a stronger promoter.
  • Animal cell culture is carried out under conditions in which the phosphate concentration and / or potassium concentration in the medium is increased. Culturing of animal cells may be carried out, for example, under conditions in which at least the concentration of phosphoric acid in the medium is increased. "Conditions in which the phosphoric acid concentration and / or potassium concentration in the medium is enhanced” are also referred to as “phosphoric acid / potassium enhancing conditions”. The “condition for enhancing the phosphoric acid concentration in the medium” is also particularly referred to as the “phosphoric acid enhancing condition”. "Conditions in which the potassium concentration in the medium is enhanced” are also particularly referred to as “potassium-enhanced conditions".
  • Control conditions include general conditions used for culturing animal cells. Specific examples of the control condition include conditions that do not satisfy the phosphoric acid / potassium enhancement conditions exemplified below.
  • the culture results of animal cells can be improved. Specifically, by culturing the animal cells under the phosphate / potassium enhancing condition, the culture performance of the animal cells can be improved as compared with the case where the animal cells are cultured under the control condition. Further, for example, by culturing the animal cells under the condition that both the phosphate concentration and the potassium concentration in the medium are enhanced, the animal cells are cultured under the condition that only one of the phosphate concentration and the potassium concentration in the medium is enhanced. The culture performance of animal cells may be improved as compared with the case. Improvements in the culture results of animal cells include improvement of animal cell proliferation, improvement of animal cell viability, improvement of target substance production by animal cells, and extension of animal cell culture period.
  • culturing animal cells under phosphate / potassium-enhanced conditions may, for example, improve the proliferation and / or viability of the animal cells.
  • culturing animal cells under phosphate / potassium-enhanced conditions may improve the proliferation and / or viability of the animal cells as compared to culturing the animal cells under control conditions.
  • the viable cell density of the animal cells is increased during culturing, for example, 1.5 times or more, 2 times or more, 2.5 times or more the viable cell density at the start of culturing.
  • the density may reach times or more, 80 times or more, 90 times or more, 100 times or more, 200 times or more, 500 times or more, or 1000 times or more.
  • the viable cell density of the animal cells can be increased, for example, 1.5 ⁇ 10 7 cells / mL or more and 1.6 ⁇ 10 7 cells / mL or more during the culture.
  • the density may reach 5 ⁇ 10 7 cells / mL or more, 7 ⁇ 10 7 cells / mL or more, 1 ⁇ 10 8 cells / mL or more, or 1 ⁇ 10 9 cells / mL or more.
  • the viable cell count of animal cells can be measured, for example, using a live-and-dead cell autoanalyzer Vi-CELL TM XR (Beckman Coulter) or a flow cytometer guava easy Cyte (Luminex).
  • the animal cell has a target substance-producing ability such as a target protein-producing ability
  • culturing the animal cell under the phosphate / potassium-enhanced condition improves, for example, the production of the target substance such as the target protein by the animal cell.
  • the animal cell has a target substance-producing ability such as a target protein-producing ability
  • the animal cell is cultured under the phosphate / potassium-enhanced condition as compared with the case where the animal cell is cultured under the control condition.
  • the production of target substances such as target proteins by animal cells may be improved.
  • the culture period of animal cells may be extended by culturing animal cells under phosphate / potassium enhancing conditions. Specifically, by culturing the animal cells under the phosphate / potassium-enhanced condition, the culture period of the animal cells may be extended as compared with the case where the animal cells are cultured under the control condition.
  • the extension of the culture period of the animal cells includes the extension of the period during which the growth of the animal cells continues, the extension of the period during which the animal cells are maintained (that is, the survival of the animal cells), and the extension of the target substance such as the target protein by the animal cells. There is an extension of the period during which production continues.
  • one aspect of the method of the present invention may be a method of improving the proliferation of animal cells.
  • one aspect of the method of the present invention may be a method for improving the survival rate of animal cells.
  • one aspect of the method of the present invention may be a method for improving the production of a target substance by an animal cell.
  • one aspect of the method of the present invention may be a method of extending the culture period of animal cells.
  • “Culture of animal cells” is not limited to those intended for the proliferation of animal cells, but also includes those not intended for the proliferation of animal cells, such as maintenance of animal cells and production of target substances by animal cells. Good.
  • the medium composition and culture conditions are not particularly limited as long as the purpose of culturing animal cells can be achieved, except that the phosphoric acid concentration and / or potassium concentration in the medium is enhanced.
  • the medium composition and culture conditions can be configured so that animal cells proliferate.
  • the purpose is to maintain animal cells
  • the medium composition and culture conditions can be configured so that the animal cells are maintained (that is, the animal cells survive).
  • the purpose is to produce a target substance such as a target protein
  • the medium composition and culture conditions can be configured so that the target substance is produced (for example, the target protein is expressed). If not intended for the growth of animal cells, the animal cells may or may not grow during culturing.
  • the animal cells may proliferate during culturing, even if they are not intended for the growth of the animal cells.
  • the medium composition and culture conditions can be appropriately set according to various conditions such as the type of animal cells.
  • the culture is carried out using, for example, the normal medium and normal conditions used for culturing animal cells as they are or with appropriate modifications, except that the phosphoric acid concentration and / or potassium concentration in the medium is enhanced. can do.
  • Culturing can be carried out using, for example, a liquid medium.
  • Specific media that can be used for culturing animal cells include D-MEM (Dulbecco's Modified Eagle Medium), CELLiST Basal Media BASAL4P (Ajinomoto Co., Ltd.), Opti-MEM (Thermo Fisher Scientific), and RPMI 1640 (Thermo Fisher Scientific).
  • the medium contains various medium components such as carbon source, amino acid, vitamin, inorganic salt, phosphoric acid, choline, amine, pH buffer, growth factor, serum, serum albumin, selective drug, gene expression inducer and the like. You can. Glucose is mentioned as a carbon source.
  • amino acids examples include 20 types of amino acids constituting proteins and their derivatives.
  • the amino acid may be, for example, L-form.
  • Specific examples of amino acids include glutamine and serine.
  • glutamine examples include L-glutamine.
  • Examples of serine include L-serine.
  • Amines include 1,4-butanediamine (also called "putresin"), agmatin, methylamine, dimethylamine, trimethylamine, ethylamine, diethylamine, triethylamine, ethylenediamine, ethanolamine, diethanolamine, triethanolamine, N, N-diisopropyl.
  • Examples thereof include ethylamine, tetramethylethylenediamine, hexamethylenediamine, spermidine, spermin and amantazine.
  • aromatic amines include aniline, phenethylamine, toluidine, catecholamines, and 1,8-bis (dimethylamino) naphthalene.
  • Heterocyclic amines include pyrrolidine, piperidine, piperidine, morpholin, quinuclidine, 1,4-diazabicyclo [2.2.2] octane, pyrrole, pyrazole, imidazole, pyridine, pyridazine, pyrimidine, pyrazine, oxazole, thiazole, 4-dimethyl.
  • Aminopyridine can be mentioned.
  • Amines include, among others, 1,4-butanediamine, ethanolamine, spermidine, spermine. Amines include, more particularly, 1,4-butanediamine.
  • any component that can form a salt may be used as a free form, as a salt, or as a combination thereof. That is, for example, the term “amino acid” may mean a free amino acid, a salt thereof, or a combination thereof, unless otherwise specified. Further, for example, the term “phosphoric acid” may mean free phosphoric acid, or a salt thereof, or a combination thereof, unless otherwise specified. Also, for example, the term “choline” may mean free choline, or a salt thereof, or a combination thereof, unless otherwise specified. Further, for example, the term “amine” may mean a free amine, a salt thereof, or a combination thereof, unless otherwise specified.
  • the salt is not particularly limited as long as it can be used for culturing animal cells.
  • a salt for an acidic group such as a phosphoric acid group or a carboxyl group
  • a salt with an alkali metal such as ammonium salt, sodium and potassium
  • a salt with an alkaline earth metal such as calcium and magnesium
  • an amine as exemplified above.
  • salts with basic amino acids such as arginine and lysine.
  • a salt for a basic group such as an amino group
  • a salt with an inorganic acid such as hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, hydrobromic acid, acetic acid, citric acid, benzoic acid, maleic acid and fumaric acid.
  • Salts with organic carboxylic acids such as tartaric acid, succinic acid, tannic acid, butyric acid, hibenzic acid, pamoic acid, enanthic acid, decanoic acid, theocric acid, salicylic acid, lactic acid, oxalic acid, mandelic acid, malic acid, methanesulfonic acid , Benzylsulfonic acid, p-toluenesulfonic acid and other salts with organic sulfonic acids.
  • examples of the salt of phosphoric acid include sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate.
  • choline salt includes choline bitartrate.
  • the seeding amount of animal cells at the start of culture is, for example, 1 ⁇ 10 3 cells / mL or more, 1 ⁇ 10 4 cells / mL or more, 1 ⁇ 10 5 cells / mL or more, 1 ⁇ 10 6 cells / mL or more, or It may be 1 ⁇ 10 7 cells / mL or more, 1 ⁇ 10 10 cells / mL or less, 1 ⁇ 10 9 cells / mL or less, 1 ⁇ 10 8 cells / mL or less, 1 ⁇ 10 7 cells / mL or less, It may be 1 ⁇ 10 6 cells / mL or less, 1 ⁇ 10 5 cells / mL or less, or 1 ⁇ 10 4 cells / mL or less.
  • the culture may be divided into a seed culture and a main culture.
  • the culture conditions of the seed culture and the main culture may or may not be the same.
  • the target substance may be produced at least during the main culture period.
  • animal cells may be sufficiently proliferated by seed culture, and then a target substance such as a target protein may be produced by main culture.
  • Culturing can be carried out by batch culture, fed-batch culture, continuous culture, or a combination thereof.
  • fed-batch culture or continuous culture include perfusion culture.
  • the medium at the start of culturing is also referred to as "initial medium” or “basal medium”.
  • the medium supplied to the culture system (for example, the initial medium) in the fed-batch culture or the continuous culture is also referred to as "fed-batch medium”.
  • supplying a fed-batch medium to a culture system in fed-batch culture or continuous culture is also referred to as "fed-batch".
  • Fed-batch may be carried out throughout the period of the culture or only for a part of the period of the culture. Fed-batch may be carried out continuously or intermittently.
  • withdrawal of the culture broth may be carried out.
  • the withdrawal of the culture solution may be, for example, withdrawal of the culture solution containing animal cells or withdrawal of the culture supernatant.
  • animal cells may be collected from the extracted culture medium and returned to the culture system.
  • Withdrawal of the culture medium may be carried out throughout the whole period of the culture, or may be carried out only during a part of the culture.
  • the withdrawal of the culture solution may be carried out continuously or intermittently.
  • the withdrawal and feeding of the culture medium may or may not be performed at the same time.
  • the amount of the culture solution to be withdrawn is preferably the same as the amount of the fed-batch medium to be fed.
  • the “equivalent amount” may mean, for example, an amount of 93 to 107% (v / v) with respect to the amount of fed-batch medium to be fed.
  • the culture forms of the seed culture and the main culture may or may not be the same.
  • Various components such as phosphoric acid may be contained in the initial medium, the fed-batch medium, or both. That is, in the process of culturing, various components such as phosphoric acid may be supplied to the medium alone or in any combination. All of these components may be supplied once or multiple times, or may be supplied continuously.
  • the composition of the initial medium and the fed-batch medium eg, the type and / or concentration of the components contained
  • the composition of the initial medium and the fed-batch medium may or may not be the same. That is, the type of the component contained in the initial medium may or may not be the same as the type of the component contained in the fed-batch medium. Further, the concentration of each component contained in the initial medium may or may not be the same as the concentration of each component contained in the fed-batch medium.
  • the composition of the initial medium and the fed-batch medium may be the same.
  • two or more fed-batch media having different compositions may be used.
  • the composition of each fed-batch medium may or may not be the same.
  • various components such as phosphoric acid may be supplied to the medium in a form such as powder that is not contained in the fed-batch medium.
  • the culture may be carried out in a CO 2 containing atmosphere such as 5% CO 2.
  • the pH of the medium may be, for example, near neutral. "Near neutral” may mean, for example, pH 6-8, pH 6.5-7.5, or pH 6.8-7.2.
  • the pH of the medium can be adjusted as needed.
  • the pH of the medium is various alkaline or acidic substances such as ammonia gas, aqueous ammonia, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, etc. Can be adjusted using.
  • the culture temperature may be, for example, 36-38 ° C.
  • the culture period is, for example, 0.5 days or more, 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 7 days or more, 8 days or more, 9 days or more, 10 days. More than 12 days, more than 15 days, or more than 20 days, 50 days or less, 40 days or less, 30 days or less, 25 days or less, 20 days or less, 15 days or less, 12 days or less, 10 days Hereinafter, it may be 9 days or less, 8 days or less, or 7 days or less, and may be a consistent combination thereof. Specifically, the culture period may be, for example, 1 to 30 days, 3 to 25 days, or 5 to 20 days. At the time of culturing, gene expression such as expression of the target protein may be induced as appropriate.
  • Various components such as phosphoric acid may be present in the medium in any form.
  • a component is a component that can be ionized, the component may or may not be ionized in the medium.
  • Concentration of a certain component may mean the total concentration of the dissolved component when the component can be present in a liquid in a plurality of forms. That is, for example, "concentration of a certain component” may mean the sum of the concentration of the molecule and the concentration of ions of the component when the component can be present in the liquid in the form of molecules and ions.
  • concentration of a component in a medium may mean the total concentration of the component dissolved in the medium when the component can be present in the medium in multiple forms. That is, for example, "concentration of a certain component in the medium” means the sum of the concentration of the molecule of the component and the concentration of ions in the medium when the component can be present in the medium in the form of molecules and ions. May mean.
  • phosphoric acid concentration may mean the total concentration of dissolved phosphoric acid molecular species. That is, the “phosphoric acid concentration", specifically, the concentration of molecular phosphoric acid (H 3 PO 4) and phosphoric acid ions (PO 4 3-, HPO 4 2- , and H 2 PO 4 -) concentration May mean the sum of.
  • phosphoric acid concentration in the medium may mean the total concentration of phosphoric acid molecular species dissolved in the medium. That is, for example, the "phosphoric acid concentration in the medium”, specifically, phosphoric acid ions in the concentration and medium phosphate molecules in the medium (H 3 PO 4) (PO 4 3-, HPO 4 2 -, and H 2 PO 4 -) may refer to the total concentration of.
  • the degree of enhancement of the phosphoric acid concentration and / or potassium concentration in the medium is not particularly limited as long as the culture performance of the animal cells (for example, the proliferation of the animal cells or the production of the target substance by the animal cells) is improved.
  • the degree of enhancement of the phosphoric acid concentration and / or potassium concentration in the medium can be appropriately set according to various conditions such as the type of animal cells, the length of the culture period, and the desired production amount of the target substance.
  • “Culturing is carried out under conditions of enhanced phosphate and / or potassium concentration in the medium” means, for example, that the phosphate and / or potassium concentration in the medium is within a predetermined range. Good.
  • the phosphoric acid concentration in the medium is, for example, 0.2 mM or more, 0.5 mM or more, 1 mM or more, 1.5 mM or more, 2 mM or more, 2.5 mM or more, 3 mM or more, 3.5 mM or more, 4 mM or more, 4.5 mM.
  • the phosphoric acid concentration in the medium may be 4 mM or more, in particular.
  • the phosphoric acid concentration in the medium may be 11 mM or more, more particularly.
  • the phosphoric acid concentration in the medium may be, for example, 0.2 to 100 mM, 4 to 70 mM, 11 to 50 mM, 11 to 40 mM, 11 to 30 mM, 11 to 27 mM, or 10 to 25 mM. ..
  • the potassium concentration in the medium is, for example, 0.2 mM or more, 0.5 mM or more, 1 mM or more, 2 mM or more, 3 mM or more, 4 mM or more, 5 mM or more, 6 mM or more, 7 mM or more, 8 mM or more, 9 mM or more, or 10 mM or more. It may be 50 mM or less, 40 mM or less, 30 mM or less, 20 mM or less, 15 mM or less, 12 mM or less, 10 mM or less, 9 mM or less, 8 mM or less, 7 mM or less, 6 mM or less, or 5 mM or less, and their contradictions.
  • the potassium concentration in the medium may be, in particular, 1 mM or higher. Specifically, the potassium concentration in the medium may be, for example, 0.2 to 50 mM, 0.5 to 30 mM, or 1 to 10 mM.
  • Phosphoric acid concentration and / or potassium concentration in the medium may be enhanced during the entire period of the culture, or may be enhanced only during a part of the culture. That is, "the culture is carried out under the condition that the phosphoric acid concentration and / or the potassium concentration in the medium is enhanced” means that the phosphoric acid concentration and / or the potassium concentration in the medium is enhanced during at least a part of the culture. It is sufficient, and it is not necessary that the phosphate concentration and / or potassium concentration in the medium is enhanced during the entire period of the culture.
  • the phosphoric acid concentration and / or potassium concentration in the medium may be increased to the above-exemplified concentration during the entire period of the culture, or may be increased to the above-exemplified concentration only during a part of the culture.
  • Phosphoric acid and / or potassium may be contained in the medium at the above-exemplified concentration at the start of culturing, or may be supplied to the medium at the above-exemplified concentration after the start of culturing. Further, phosphoric acid and / or potassium is contained in the medium at the above-exemplified concentration at the start of culturing, and is further supplied to the medium at the above-exemplified concentration after the start of culturing (for example, after consumption). You may. When the culture is divided into seed culture and main culture, the phosphate concentration and / or potassium concentration in the medium should be at least during the main culture, that is, during the entire main culture or a part of the main culture.
  • the phosphate concentration and / or potassium concentration in the medium may or may not be enhanced during the seed culture period.
  • the description about the culture for example, “culture period (culture period)” or “culture start" can be read as that for the main culture.
  • the “partial period” is not particularly limited as long as the culture performance of the animal cells (for example, the proliferation of the animal cells and the production of the target substance by the animal cells) is improved.
  • the “partial period” can be appropriately set according to various conditions such as the type of animal cells, the length of the culture period, and the desired production amount of the target substance.
  • “Partial period” is, for example, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more of the entire culture period. It may be there.
  • the "partial period” is, for example, 0.5 days or more, 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 7 days or more, 8 days or more, The period may be 9 days or more, 10 days or more, 12 days or more, or 15 days or more.
  • the phosphoric acid concentration and / or potassium concentration in the medium may be enhanced to the above-exemplified concentration, for example, as an average value over the entire culture period. That is, "culture is carried out under the condition that the phosphate concentration and / or potassium concentration in the medium is increased to a certain concentration” or "culture is carried out in a medium having a certain phosphoric acid concentration and / or potassium concentration”. By may mean that the average value of phosphoric acid concentration and / or potassium concentration in the medium throughout the whole period of the culture is within the range of the concentration.
  • the "average value of phosphoric acid concentration and / or potassium concentration in the medium throughout the entire culture period” is not particularly limited as long as the fluctuation of phosphoric acid concentration and / or potassium concentration during the entire culture period can be grasped. May mean, for example, the average value of phosphate and / or potassium concentration in the medium measured every 60 minutes, every 30 minutes, every 20 minutes, or every 10 minutes throughout the culture.
  • the culture is carried out under the condition that the concentration of phosphoric acid and / or the concentration of potassium in the medium is increased” means that, for example, the amount of phosphoric acid and / or potassium supplied to the medium is within a predetermined range. It may mean.
  • the amount of phosphoric acid supplied to the medium is, for example, 0.05 mM or more, 0.1 mM or more, 0.15 mM or more, 0.2 mM or more, 0.25 mM or more, 0.3 mM or more per day throughout the culture.
  • 0.35 mM or more 0.35 mM or more, 0.4 mM or more, 0.45 mM or more, 0.5 mM or more, 0.55 mM or more, 0.6 mM or more, 0.65 mM or more, 0.7 mM or more, 0.75 mM or more, 0.8 mM or more , 0.85 mM or more, 0.9 mM or more, 0.95 mM or more, or 1 mM or more, 5 mM or less, 4 mM or less, 3 mM or less, 2 mM or less, or 1 mM or less, and there is no contradiction between them. It may be a combination.
  • the amount of phosphoric acid supplied to the medium may be 0.05 mM or more per day, especially throughout the culture.
  • the amount of phosphoric acid supplied to the medium may be 0.5 mM or more per day, more particularly throughout the entire culture.
  • the amount of phosphoric acid supplied to the medium is, for example, 0.05 to 5 mM, 0.1 to 4 mM, 0.4 to 3 mM, or 0.7 to 2 mM per day throughout the culture. It may be.
  • the amount of potassium supplied to the medium is, for example, 0.05 mM or more, 0.1 mM or more, 0.15 mM or more, 0.2 mM or more, 0.25 mM or more, 0.3 mM or more per day throughout the culture.
  • the amount of potassium supplied to the medium may be 0.2 mM or more per day, especially throughout the culture. Specifically, the amount of potassium supplied to the medium may be, for example, 0.05 to 5 mM, 0.1 to 3 mM, or 0.2 to 1 mM per day throughout the culture.
  • Phosphoric acid and / or potassium may be continuously supplied to the medium or intermittently supplied to the medium, for example. Phosphoric acid and / or potassium may be supplied to the medium daily or every few days, for example.
  • Supply of phosphoric acid and / or potassium to the medium for example, viable cell density of the animal cells 1.5 ⁇ 10 6 cells / mL or more, 1.6 ⁇ 10 6 cells / mL or more, 1.7 ⁇ 10 6 cells / mL or more, 1.8 ⁇ 10 6 cells / mL or more, 1.9 ⁇ 10 6 cells / mL or more, 2 ⁇ 10 6 cells / mL or more, 3 ⁇ 10 6 cells / mL or more, 5 ⁇ 10 6 cells It may be carried out at a density of / mL or higher, 7 ⁇ 10 6 cells / mL or higher, 1 ⁇ 10 7 cells / mL or higher, or 1 ⁇ 10 8 cells / mL or higher.
  • Culturing may be carried out in the presence of amines, choline, and / or serins. Culturing may be carried out, for example, in the presence of amines, choline, and / or serins, at least when the phosphate concentration in the medium is enhanced. Culturing may be carried out, for example, in the presence of at least amine. Culturing may be carried out, for example, in the presence of at least choline and / or serin.
  • “Culture is performed in the presence of amines, choline, and / or serin” may mean, for example, that the medium contains amines, choline, and / or serine.
  • the amine concentration in the medium is, for example, 0.001 mM or more, 0.002 mM or more, 0.005 mM or more, 0.01 mM or more, 0.02 mM or more, 0.03 mM or more, 0.04 mM or more, 0.05 mM or more, 0.
  • the amine concentration in the medium may be, in particular, 0.002 mM or more.
  • the amine concentration in the medium may be, more particularly, 0.007 mM or more.
  • the amine concentration in the medium may be, for example, 0.002-0.5 mM, 0.005-0.2 mM, or 0.007-0.1 mM.
  • the choline concentration in the medium is, for example, 0.1 mM or more, 0.2 mM or more, 0.5 mM or more, 1 mM or more, 2 mM or more, 3 mM or more, 4 mM or more, 5 mM or more, 6 mM or more, 7 mM or more, 8 mM or more, 9 mM or more.
  • the choline concentration in the medium may be 0.2 mM or more, in particular.
  • the choline concentration in the medium may be, more particularly, 1 mM or more.
  • the choline concentration in the medium may be, for example, 0.2 to 50 mM, 0.5 to 20 mM, or 1 to 10 mM.
  • the serine concentration in the medium is, for example, 0.5 mM or more, 1 mM or more, 2 mM or more, 3 mM or more, 4 mM or more, 5 mM or more, 6 mM or more, 7 mM or more, 8 mM or more, 9 mM or more, 10 mM or more, 12 mM or more, 15 mM or more, It may be 20 mM or more, 25 mM or more, 30 mM or more, 35 mM or more, 40 mM or more, 50 mM or more, 60 mM or more, 70 mM or more, 80 mM or more, 90 mM or more, or 100 mM or more, 500 mM or less, 400 mM or less, 300 mM or less, 200 mM.
  • the serine concentration in the medium may be particularly 2 mM or more.
  • the serine concentration in the medium may be more than 10 mM, more particularly.
  • the serine concentration in the medium may be, for example, 1 to 200 mM, 2 to 100 mM, 4 to 50 mM, 2 to 500 mM, 5 to 200 mM, or 10 to 100 mM.
  • Amine, choline, and / or serine may be contained in the medium for the entire period of the culture, or may be contained in the medium only for a part of the period of the culture. That is, "culture is performed in the presence of amines, choline, and / or serine" or "culture is performed in a medium containing amine, choline, and / or serine” means that amine, choline, and It is sufficient that / or serine is contained in the medium for at least a part of the culture, and it is not necessary that amine, choline, and / or serine be contained in the medium for the entire duration of the culture.
  • Amine, choline, and / or serine may be contained in the medium at the above-exemplified concentrations, for example, during the entire period of the culture, and may be contained in the medium at the above-exemplified concentrations only during a part of the culture. May be good. That is, "culture is carried out in the presence of certain amine, choline and / or serine" or "culture is carried out in medium of certain amine, choline and / or serine concentration".
  • the amine concentration, choline concentration, and / or serine concentration in the medium is within the range of the concentration during at least a part of the culture, and the amine concentration, choline concentration, and / or serine concentration in the medium is It does not have to be within the concentration range for the entire duration of the culture.
  • the amine concentration, choline concentration, and / or serine concentration in the medium may be set to the above-exemplified concentration, for example, as an average value over the entire culture period. That is, "culture is carried out in the presence of certain amine, choline, and / or serine” or "culture is carried out in medium of certain amine, choline, and / or serine concentrations.” May mean that the average value of amine, choline, and / or serine concentrations in the medium throughout the culture is within that concentration range.
  • the "average value of amine concentration, choline concentration, and / or serine concentration in the medium throughout the entire culture period” means that the fluctuation of amine concentration, choline concentration, and / or serine concentration during the entire culture period can be grasped. This is not particularly limited, but for example, the amine concentration, choline concentration, and / or serine concentration in the medium measured every 60 minutes, 30 minutes, 20 minutes, or 10 minutes throughout the culture period. It may mean an average value.
  • culture is carried out in the presence of amines, choline, and / or serin may mean, for example, that the medium is supplied with amine, choline, and / or serine.
  • the amount of amine supplied to the medium is, for example, 0.0002 mM or more, 0.0005 mM or more, 0.001 mM or more, 0.0015 mM or more, 0.002 mM or more, 0.0025 mM or more, per day throughout the culture. It may be 0.003 mM or more, 0.0035 mM or more, 0.004 mM or more, 0.0045 mM or more, or 0.005 mM or more, 0.025 mM or less, 0.02 mM or less, 0.015 mM or less, 0.01 mM or less.
  • the supply of amine to the medium may be 0.0005 mM or more per day, especially throughout the culture.
  • the amount of amine supplied to the medium is, for example, 0.0005 to 0.025 mM mM, 0.001 to 0.015 mM, 0.002 to 0.007 mM, or 0.003 to 0 per day throughout the culture. It may be .004 mM.
  • the amount of choline supplied to the medium is, for example, 0.02 mM or more, 0.05 mM or more, 0.1 mM or more, 0.15 mM or more, 0.2 mM or more, 0.25 mM or more per day throughout the culture. It may be 0.3 mM or more, 0.35 mM or more, 0.4 mM or more, 0.45 mM or more, or 0.5 mM or more, 2.5 mM or less, 2 mM or less, 1.5 mM or less, 1 mM or less, 0.7 mM. Hereinafter, it may be 0.5 mM or less, 0.2 mM or less, or 0.1 mM or less, and may be a consistent combination thereof.
  • the supply of choline to the medium may be 0.05 mM or more per day, especially throughout the culture.
  • the supply of choline to the medium is, for example, 0.05-2.5 mM, 0.1-1.5 mM, 0.2-0.7 mM, or 0.3-0 per day throughout the culture. It may be .5 mM.
  • the amount of serin supplied to the medium is, for example, 0.1 mM or more, 0.2 mM or more, 0.3 mM or more, 0.4 mM or more, 0.5 mM or more, 0.6 mM or more, per day throughout the culture.
  • the supply of serin to the medium may be 0.5 mM or more per day, especially throughout the culture.
  • the amount of serin supplied to the medium is, for example, 0.2 to 10 mM, 0.4 to 5 mM, 0.8 to 3 mM, 0.5 to 25 mM, 1 to 15 mM, 2 to per day throughout the culture. It may be 7 mM, or 3.5-5 mM.
  • Amine, choline, and / or serine may be supplied to the medium continuously or intermittently, for example. Amines, choline, and / or serine may be supplied to the medium daily or every few days, for example.
  • the phosphoric acid concentration in the fed-batch medium is not particularly limited as long as the desired amount of phosphoric acid can be supplied.
  • the phosphoric acid concentration in the fed-batch medium may be, for example, the phosphoric acid concentration in the above-exemplified medium.
  • the phosphoric acid concentration in the fed-batch medium may be the phosphoric acid concentration in the above-exemplified medium.
  • the phosphoric acid concentration in the fed-batch medium is, for example, more than 1 times, 1.1 times or more, 1.3 times or more, 1.5 times or more, 2 times or more the phosphoric acid concentration in the above-exemplified medium.
  • the concentration may be 3 times or more, 5 times or more, 7 times or more, 10 times or more, 15 times or more, or 20 times or more, and 100 times or less, 70 times or less, 50 times or less, 30 times or less, 20 times or more.
  • the concentration may be 10 times or less, or 5 times or less, and may be a consistent combination thereof.
  • the phosphoric acid concentration in the fed-batch medium may be, for example, 10 mM or more, 20 mM or more, 30 mM or more, 50 mM or more, 70 mM or more, 100 mM or more, 150 mM or more, or 200 mM or more, 1000 mM or less, 700 mM or less, It may be 500 mM or less, 300 mM or less, 200 mM or less, 100 mM or less, 70 mM or less, or 50 mM or less, and may be a consistent combination thereof. Specifically, the phosphoric acid concentration in the fed-batch medium may be, for example, 10 to 1000 mM, 30 to 500 mM, or 50 to 200 mM.
  • the amine concentration in the fed-batch medium is not particularly limited as long as the desired amount of amine can be supplied.
  • the amine concentration in the fed-batch medium may be, for example, the amine concentration in the above-exemplified medium.
  • the amine concentration in the fed-batch medium may be the amine concentration in the above-exemplified medium.
  • the amine concentration in the fed-batch medium is, for example, more than 1 times, 1.1 times or more, 1.3 times or more, 1.5 times or more, 2 times or more, or 3 times the amine concentration in the above-exemplified medium.
  • the concentration may be 5 times or more, 7 times or more, 10 times or more, 15 times or more, or 20 times or more, 100 times or less, 70 times or less, 50 times or less, 30 times or less, 20 times or less,
  • the concentration may be 10 times or less, or 5 times or less, and may be a consistent combination thereof.
  • the amine concentration in the fed-batch medium is, for example, 0.05 mM or more, 0.1 mM or more, 0.2 mM or more, 0.3 mM or more, 0.5 mM or more, 0.7 mM or more, 1 mM or more, or 1 mM or more.
  • the amine concentration in the fed-batch medium may be, for example, 0.05 to 5 mM, 0.1 to 3 mM, or 0.2 to 1 mM.
  • the choline concentration in the fed-batch medium is not particularly limited as long as the desired amount of choline can be supplied.
  • the choline concentration in the fed-batch medium may be, for example, the choline concentration in the above-exemplified medium.
  • the choline concentration in the fed-batch medium may be the choline concentration in the above-exemplified medium.
  • the choline concentration in the fed-batch medium is, for example, more than 1 times, 1.1 times or more, 1.3 times or more, 1.5 times or more, 2 times or more, and 3 times the choline concentration in the above-exemplified medium.
  • the concentration may be 5 times or more, 7 times or more, 10 times or more, 15 times or more, or 20 times or more, 100 times or less, 70 times or less, 50 times or less, 30 times or less, 20 times or less,
  • the concentration may be 10 times or less, or 5 times or less, and may be a consistent combination thereof.
  • the choline concentration in the fed-batch medium may be, for example, 5 mM or more, 10 mM or more, 20 mM or more, 30 mM or more, 50 mM or more, 70 mM or more, or 100 mM or more, and 500 mM or less, 300 mM or less, 200 mM or less, 100 mM.
  • the choline concentration in the fed-batch medium may be, for example, 5 to 500 mM, 10 to 300 mM, or 20 to 100 mM.
  • the serine concentration in the fed-batch medium is not particularly limited as long as the desired amount of serine to be supplied can be obtained.
  • the serine concentration in the fed-batch medium may be, for example, the serine concentration in the above-exemplified medium.
  • the serine concentration in the fed-batch medium may be the serine concentration in the above-exemplified medium.
  • the serine concentration in the fed-batch medium is, for example, more than 1 times, 1.1 times or more, 1.3 times or more, 1.5 times or more, 2 times or more and 3 times the serine concentration in the above-exemplified medium.
  • the concentration may be 5 times or more, 7 times or more, 10 times or more, 15 times or more, or 20 times or more, 100 times or less, 70 times or less, 50 times or less, 30 times or less, 20 times or less, The concentration may be 10 times or less, or 5 times or less, and may be a consistent combination thereof.
  • the serine concentration in the fed-batch medium may be, for example, 20 mM or more, 30 mM or more, 50 mM or more, 70 mM or more, 100 mM or more, 200 mM or more, 300 mM or more, 500 mM or more, 700 mM or more, or 1000 mM or more, and 5000 mM.
  • the serine concentration in the fed-batch medium may be, for example, 30 to 2000 mM, 50 to 1000 mM, 70 to 500 mM, 50 to 5000 mM, 100 to 3000 mM, or 200 to 1000 mM.
  • the concentration of various components can be measured, for example, by a known method used for detecting or identifying a compound. Such methods include, for example, HPLC, UPLC, LC / MS, GC / MS, NMR. These methods can also be used to confirm that the target substance has been produced. One of these methods may be used alone, or two or more thereof may be used in combination as appropriate.
  • Animal cells can be cultured as described above.
  • the target substance is produced (for example, the target protein is expressed) by culturing the animal cell as described above, and thus the target substance is contained.
  • the culture to be used is obtained.
  • the target substance such as the target protein may be accumulated in the medium, the cell surface, the cell, or a combination thereof.
  • the production of the target protein can be confirmed by a known method used for protein detection or identification.
  • a known method used for protein detection or identification examples include SDS-PAGE, Western blotting, mass spectrometry, N-terminal amino acid sequence analysis, and enzyme activity measurement.
  • SDS-PAGE SDS-PAGE
  • Western blotting Western blotting
  • mass spectrometry mass spectrometry
  • N-terminal amino acid sequence analysis N-terminal amino acid sequence analysis
  • enzyme activity measurement One of these methods may be used alone, or two or more thereof may be used in combination as appropriate.
  • the target protein can be recovered as appropriate. Specifically, the target protein can be recovered as an appropriate fraction containing the target protein. Examples of such a fraction include cultures, culture supernatants, cultured cells, and processed products of cultured cells (crushed products, lysates, extracts (cell-free extracts). Cultured cells include, for example, It may be obtained in the form of immobilized cells immobilized with a carrier such as acrylamide or carrageenan.
  • the target protein may be further purified to the desired degree.
  • the target protein When the target protein accumulates in the medium, the target protein can be purified from the supernatant after removing the solid content of cells or the like from the culture by centrifugation or the like.
  • the target protein When the target protein accumulates in the cell, the target protein can be purified from the processed product after being subjected to a treatment such as crushing, lysing, or extracting the cell, for example.
  • the cells can be recovered from the culture by centrifugation or the like.
  • Treatments such as cell disruption, lysis, and extraction can be performed by known methods. Examples of such a method include an ultrasonic crushing method, a dynomill method, a bead crushing method, a French press crushing method, and a lysozyme treatment. One of these methods may be used alone, or two or more thereof may be used in combination as appropriate.
  • the target protein When the target protein accumulates on the cell surface, the target protein can be purified from the solubilized product after being solubilized, for example.
  • Solubilization can be performed by a known method. Such methods include, for example, increasing the salt concentration and using a surfactant. One of these methods may be used alone, or two or more thereof may be used in combination as appropriate.
  • Purification of the target protein can be performed by a known method used for protein purification.
  • a known method used for protein purification include sulfur fractionation, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, gel filtration chromatography, and isoelectric point precipitation.
  • sulfur fractionation ion exchange chromatography
  • hydrophobic chromatography affinity chromatography
  • gel filtration chromatography gel filtration chromatography
  • isoelectric point precipitation One of these methods may be used alone, or two or more thereof may be used in combination as appropriate.
  • the target protein may be obtained in a free state or in a state of an immobilized enzyme immobilized on a solid phase such as a resin.
  • the recovered target protein may be appropriately formulated.
  • the dosage form is not particularly limited and can be appropriately set according to various conditions such as the intended use of the target protein. Dosage forms include, for example, liquids, suspensions, powders, tablets, pills and capsules. In formulation, pharmacologically acceptable additives such as excipients, binders, disintegrants, lubricants, stabilizers, flavoring agents, odorants, fragrances, diluents, surfactants, etc. Can be used.
  • the medium of the present invention is a medium for culturing animal cells in which the phosphoric acid concentration and / or the potassium concentration is enhanced.
  • the medium of the present invention can be used, for example, in the method of the present invention.
  • the description of the medium used in the method of the present invention can be applied mutatis mutandis.
  • the medium of the present invention contains phosphoric acid and / or potassium.
  • the medium of the present invention may further contain amines, choline, and / or serine.
  • the medium of the present invention may contain amines, choline, and / or serine, for example, if it contains at least phosphoric acid.
  • the medium of the present invention may contain, for example, at least an amine.
  • the medium of the present invention may contain, for example, at least choline and / or serine.
  • the medium of the present invention may be, for example, a basal medium (initial medium) or a fed-batch medium.
  • the fed-batch medium may be used for, for example, fed-batch culture or continuous culture.
  • the fed-batch medium may be, for example, one used for perfusion culture.
  • the description of the phosphoric acid concentration in the medium (including the description of the phosphoric acid concentration in the feed medium) in the method of the present invention exemplified above can be applied mutatis mutandis.
  • the description of the potassium concentration in the medium (including the description of the potassium concentration in the fed-batch medium) in the method of the present invention exemplified above can be applied mutatis mutandis.
  • the description of the amine concentration in the medium (including the description of the amine concentration in the fed-batch medium) in the method of the present invention exemplified above can be applied mutatis mutandis.
  • the description of the choline concentration in the medium (including the description of the choline concentration in the fed-batch medium) in the method of the present invention exemplified above can be applied mutatis mutandis.
  • the description of the serine concentration in the medium (including the description of the serine concentration in the feed medium) in the method of the present invention exemplified above can be applied mutatis mutandis.
  • Example 1 Evaluation of the effect of improving CHO cell proliferation and antibody production by adding phosphoric acid to the feeding medium
  • the effect of improving CHO cell proliferation and antibody production by adding phosphoric acid to the feeding medium was evaluated. evaluated.
  • CHO cells cells of the CHO DG-44 lineage modified to produce the antibody Adalimumab were used.
  • Basal medium CELLiST registered trademark
  • Basal media (Ajinomoto Co., Ltd .: BASAL4P; containing phosphoric acid with a final concentration of 1.9 mM), LR3-IGF-1 (final concentration 10 ⁇ g / mL), sodium dextran sulfate (Final concentration 400 mg / L) and L-Glutamine (final concentration 6 mM) were added to prepare a basal medium.
  • Feed media (Ajinomoto Co., Ltd .: FEED2)
  • glucose final concentration 75 g / L
  • sodium dihydrogen phosphate final concentration 0, 125, 250, 500, 1000
  • 4000 or 7000 mg / L (ie, 0.0, 1.0, 2.1, 4.2, 8.3, 33.3, or 58.3 mM) was added to prepare seven fed-batch media with different phosphate concentrations.
  • Cell culture medium was sampled on days 4, 7, 9, 11, and 14 of the start of culture, and the number of viable cells was measured using a living-and-dead cell autoanalyzer Vi-CELL TM XR (Beckman Coulter), and the amount of antibody produced. was measured using Octet QK (FORTEBIO).
  • Example 2 Evaluation of the effect of improving CHO cell proliferation and antibody production by adding phosphoric acid to the feed medium
  • the effect of improving CHO cell proliferation and antibody production by adding phosphoric acid to the feed medium was evaluated. evaluated.
  • CHO cells cells of the CHO DG-44 lineage modified to produce the antibody Adalimumab were used.
  • Basal medium CELLiST registered trademark
  • Basal media (Ajinomoto Co., Ltd .: BASAL4P; containing phosphoric acid with a final concentration of 1.9 mM), LR3-IGF-1 (final concentration 10 ⁇ g / mL), diphosphate Sodium hydrogen hydrogen (115 mg / L (ie, final concentration 2.9 mM)), sodium dextran sulfate (final concentration 400 mg / L) and L-Glutamine (final concentration 6 mM) were added to prepare a basal medium.
  • Basal media (Ajinomoto Co., Ltd .: BASAL4P; containing phosphoric acid with a final concentration of 1.9 mM
  • LR3-IGF-1 final concentration 10 ⁇ g / mL
  • diphosphate Sodium hydrogen hydrogen 115 mg / L (ie, final concentration 2.9 mM)
  • sodium dextran sulfate final concentration 400 mg / L
  • L-Glutamine final concentration
  • Feed media (Ajinomoto Co., Ltd .: FEED2)
  • glucose final concentration 75 g / L
  • sodium dihydrogen phosphate final concentration 0, 1000, 2000, 3000, 4000
  • 5000 mg / L ie 0.0, 8.3, 16.7, 25, 33.3, or 41.7 mM
  • Cell culture medium was sampled on days 4, 7, 9, 11, and 14 of the start of culture, and the number of viable cells was measured using a living-and-dead cell autoanalyzer Vi-CELL TM XR (Beckman Coulter), and the amount of antibody produced. was measured using CEDEX Bio HT (Roche Diagnostics).
  • Example 3 Evaluation of the effect of adding phosphoric acid and serine, choline, or amine to the feed medium to improve CHO cell proliferation and antibody production
  • phosphoric acid and serine, choline, etc. to the feed medium
  • the effect of adding amine on improving CHO cell proliferation and antibody production was evaluated.
  • CHO cells cells of the CHO S lineage modified to produce the antibody Adalimumab were used.
  • Basal medium CELLiST registered trademark
  • Basal media (Ajinomoto Co., Ltd .: BASAL4P; containing phosphoric acid with a final concentration of 1.9 mM), LR3-IGF-1 (final concentration 10 ⁇ g / mL), diphosphate Sodium hydrogen hydrogen (115 mg / L (ie, total final concentration 2.9 mM with phosphate contained in Basal media)), sodium dextran sulfate (final concentration 400 mg / L), and L-Glutamine (final concentration 6 mM) ) was added to prepare a basal medium.
  • Basal media Ajinomoto Co., Ltd .: BASAL4P
  • LR3-IGF-1 final concentration 10 ⁇ g / mL
  • diphosphate Sodium hydrogen hydrogen 115 mg / L (ie, total final concentration 2.9 mM with phosphate contained in Basal media)
  • sodium dextran sulfate final concentration 400 mg / L
  • Feed media (Ajinomoto Co., Ltd .: FEED2), glucose (final concentration 75 g / L), sodium dihydrogen phosphate (final concentration 0 or 2500 mg / L (ie, that is) 0.0 or 20.8 mM)), and if necessary, the following components (A), (B), (C), or (D) were added to prepare 10 types of fed-batch media: (A) 1,4-Butanediamine (final concentration 12.8 mg / L (ie 0.145 mM)); (B) Serine (final concentration 20,000 mg / L (ie 190 mM)), choline bitartrate (final concentration 4,400 mg / L (ie 17.4 mM)), and 1,4-butanediamine (final concentration 12.8 mg /).
  • Cell culture medium was sampled on days 4, 7, 9, 11 and 14 of the start of culture, the number of viable cells was measured using a flow cytometer guava easyCyte (Luminex), and the amount of antibody produced was measured by CEDEX Bio HT (Roche. It was measured using Diagnostics).
  • Luminex flow cytometer guava easyCyte
  • CEDEX Bio HT Roche. It was measured using Diagnostics.
  • Example 4 Evaluation of CHO cell proliferation and antibody production improving effect by addition of phosphoric acid or potassium to the feed medium
  • CHO cell proliferation and CHO cell proliferation by addition of phosphoric acid or potassium to the feed medium The effect of improving antibody production was evaluated.
  • CHO cells cells of the CHO S lineage modified to produce the antibody Herceptin were used.
  • Cell culture medium was sampled on days 4, 7, 9, 11, and 14 of the start of culture, and the number of viable cells was measured using a living-and-dead cell autoanalyzer Vi-CELL TM XR (Beckman Coulter), and the amount of antibody produced. was measured using CEDEX Bio HT (Roche Diagnostics).
  • cell culture medium was sampled on the 4th, 7th, 9th, and 11th days after the start of culture, and the glucose concentration was measured using CEDEX Bio HT (Roche Diagnostics), and the final glucose concentration was 11 g / A 500 g / L glucose aqueous solution was added so as to have L.
  • Example 5 Evaluation of the effect of improving antibody production by phosphate-enriched medium in Perfusion culture
  • the effect of improving CHO cell proliferation and antibody production by adding phosphate to the basal medium and flow medium was applied to Perfusion culture. Evaluated.
  • CHO cells cells of the CHO S lineage modified to produce the antibody Adalimumab were used.
  • the medium exchange was carried out by centrifuging the culture medium, removing it so that 3.68 mL of the centrifugal supernatant remained, adding 6.32 mL of the same fresh fed-batch medium as the basal medium, and stirring. After changing the medium, the culture was restarted. The number of living cells was measured using a living-and-dead cell autoanalyzer Vi-CELL TM XR (Beckman Coulter), and the amount of antibody produced was measured using CEDEX Bio HT (Roche Diagnostics).
  • Example 6 Evaluation of the effect of improving antibody production by the phosphate-enriched medium in Perfusion culture
  • the effect of improving CHO cell proliferation and antibody production by adding phosphate to the flow medium was evaluated in Perfusion culture. ..
  • CHO cells cells of the CHO S lineage modified to produce the antibody Adalimumab were used.
  • the medium exchange was carried out by centrifuging the culture medium, removing it so that 3.68 mL of the centrifugal supernatant remained, and adding 6.32 mL of fresh fed-batch medium. After changing the medium, the culture was restarted.
  • the number of living cells was measured using a living-and-dead cell autoanalyzer Vi-CELL TM XR (Beckman Coulter), and the amount of antibody produced was measured using CEDEX Bio HT (Roche Diagnostics).
  • animal cells for example, proliferation of animal cells and production of target substance by animal cells.

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See also references of EP4056678A4
SHUICHI HASHIZUME, KAZUHIKO KURODA, MASANORI KAMEI: "Generation and production of human monoclonal antibody", NIPPON NOGEIKAGAKU KAISHI, vol. 62, no. 10, 15 October 1988 (1988-10-15), pages 1513 - 1516, XP055929703, ISSN: 0002-1407, DOI: 10.1271/nogeikagaku1924.62.1513 *

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