JPH0870859A - Serum-free culture medium for culturing animal cell and production of physiologically active peptide or protein - Google Patents

Abstract

PURPOSE: To obtain an inexpensive and uniform serum-free culture medium for culturing an animal cell, containing an inorganic iron compound, etc., cyclodextrin and a nonionic surfactant in a basal culture medium without any difference between lots and useful for producing physiologically active peptides, etc. CONSTITUTION: This serum-free culture medium for culturing an animal cell contains (A) an inorganic or an organic iron compound such as ferric chloride, ferrous chloride, ferrous sulfate, iron nitrate, iron citrate, iron oxalate or iron lactate, (B) a cyclodextrin such as α-cyclodextrin and (C) a nonionic surfactant such as Pluronic (R)-based surfactant and further preferably (D) insulin, ethanolamine (derivative) and selenious acid (salt) in a basal culture medium. Furthermore, the resultant culture medium is preferably used to culture the animal cell such as a cell of a myeloma, e.g. a cell of a murine myeloma having the productivity for t-gD-IL-2 or Sp2-/0-22-32-34 strain and produce a physiologically active peptide or protein.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a serum-free medium for large-scale culture of animal cells, a method for producing a physiologically active peptide or protein using the medium, and a cell line producing the same.

[0002]

2. Description of the Related Art Large-scale culture of animal cells, especially transgenic animal cells, is carried out by using, for example, physiologically active peptides or proteins such as interferon, erythropoietin, tissue plasminogen activator, granulocyte colony stimulating factor, nerve growth factor, and the like. Antigens for vaccines against various viruses, immunoglobulin variable regions (Idiot
ype) -granulocyte / macrophage colony stimulating factor (GM
-CSF) fusion protein (Idiotype / GM-CSF) (MT
ao and R. Levy, Nature, Volume 362,
755 (1993)), t-gD-IL-2 fusion protein (S. Hinuma et al., FEBS Letters, 288, 138 (1991)). It is a very important technology for manufacturing. Host cells for producing bioactive peptides or proteins include human Burkitt lymphoma (Namalwa) cells and mouse myeloma cells (mouse myeloma Sp2 / O-
Floating cells such as Ag14 and mouse myeloma X63Ag8-653) and C derived from Chinese hamster ovary
HO cells, BHK cells derived from baby hamster kidney,
HeLa cells derived from human cervical cancer, C-derived from mouse breast cancer
Both 127 cells and adherent cells such as mouse fibroblasts (NIH / 3T3) are used, and especially CH cells are conventionally used.
O cells are widely used. For adherent cells such as CHO cells, a roller bottle culture, a microcarrier culture, or an immobilization culture method in which the cells are adhered to an immobilization bed such as hollow fiber, a ceramic matrix or glass cloth and cultured is adopted. There is. However, in order to mass-produce industrially useful substances, suspension culture is more advantageous than adhesive culture in terms of operation and scale-up. Therefore, a method of acclimatizing CHO cells so that they can be suspension-cultured (M. Murata et al., J. Ferment. Technol.
l.), 66, 501 (1988)) or a method using Namalwa cells that are naturally floating (H. Yanagi et al., Journal of Fermentation and Bioengineering (J. Ferment. Bioeng.), Volume 68,
257 (1989); M. Okamoto et al., Bio / Technology, June, 550 (199).
0)); S. Hosoi et al., Site Technology (Cytotechnol
ogy), Vol. 7, p. 25 (1991)) has been attracting attention in recent years. An expression system using a myeloma cell as a host cell is also known (JP-A-63-44897; JP-A-63-126483), but a physiologically active peptide or protein was actually produced in large quantities and efficiently using this cell. There are relatively few examples [D. Broad et al.
Technology, Vol. 5, p. 47 (1991)].

Serum is generally required for animal cells to proliferate, and it is often impossible or impossible to proliferate in a serum-free medium (hereinafter also referred to as a serum-free medium). . However, serum is expensive, has a large difference in quality between lots, and contains a large amount of proteinaceous substances whose components are unknown, and thus interferes with the purification of the produced physiologically active peptide or protein of interest. Particularly in the case of pharmaceuticals, if a medium containing serum (hereinafter sometimes referred to as a serum medium) is used, a complicated test for removing impurities derived from serum is required for safety evaluation. Therefore, when industrially producing substances using animal cells, the serum medium has many problems, and development of a practical serum-free medium is desired. So far, blood coagulation factor VIII production C
As a serum-free medium for HO cells, JP-A-4-31648
4 discloses a medium in which Pluronic F68 and dimethyl-α-cyclodextrin are added to ASF-104 medium, but when using this medium, “first, target cells should be treated with a medium containing 10% serum. Incubate for 8-24 hours,
(Page 3, right column, lines 2 to 3) ", it is necessary to pre-culture the cells in a medium containing 10% serum, and the culture is not completely serum-free. Also,
It is a culture method far from industrial substance production, only an example of small-scale static culture on a culture dish is described. On the other hand, as a serum-free medium for culturing mouse hybridoma, JP-A-4-51891 and JP-A-3
-180175 includes ITES (insulin, transferrin, ethanolamine, sodium selenite)
A medium in which an inorganic iron compound is added instead of transferrin in the medium is disclosed, but even in these cases, only examples of static culture on a small scale in a tissue culture flask are described, and However, it is unclear whether these media can be applied to the culture of CHO cells which are generally used as hosts for gene recombination. As described above, the fact is that an inexpensive and highly productive industrial serum-free medium useful for industrial production of physiologically active peptides or proteins by animal cells has not yet been provided.

By the way, in order to efficiently produce useful substances by culturing animal cells, it is generally said that a method of continuously culturing cells at a high density for a long period of time is advantageous. In fact, for culturing floating cells such as monoclonal antibody-producing hybridomas and transgenic mouse myeloma, fresh medium is supplied into the culture tank and the culture supernatant liquid containing waste products is harvested outside the culture tank for a long period of time. , A continuous high-density suspension perfusion culture method has been tried (K. Kit
ano et al., Applied Microbiology and Biotechnology (Appl. Microbiolo. Biotech.), Vol. 24, page 282 (1986); Y. Takazawa and
M. Tokashiki, Site Technology (Cytotechnolog
y), Vol. 2, p. 95 (1989)). On the other hand, the batch culturing method is excellent in reproducibility of culturing, and can be said to be a culturing method advantageous for industrial production in order to secure the homeostasis of the product. In addition, batch culture has advantages that perfusion culture requires less auxiliary equipment other than a culture tank, so that the culture process can be simplified and scale-up is easy. A fed-batch culture method, in which medium components are fed and cultured, has been attempted as a means of taking advantage of such batch culture and increasing productivity (culturing myeloma cells in serum-free medium M. Rhodes and J. Birch). , Biotechnology (B
io / Technology), Vol. 6, p. 518 (1988).

[0005]

As described above, it is desired to develop an efficient method for producing a physiologically active peptide or protein by culturing animal cells in a large amount using a serum-free medium. Is not well established.
For example, in order to produce human NT-3, a method using human NT-3 gene-introduced CHO cells is disclosed in JP-A-5-1036.
No. 75, but this method employs a method of culturing in a medium containing 5% fetal bovine serum and then changing to a commercially available serum-free medium during the culturing. In addition, the variable region (Idiotype) of immunoglobulin molecules derived from cancer cells and granulocyte / macrophage colony stimulating factor (GM-CSF)
Research on expressing fusion protein (Idiotype / GM-CSF) with animal cells in animal cells and using it as a vaccine against B-cell lymphoma (M. Tao and R. Levy, Nature, Volume 362, Vol. Page 755 (199
3)), the animal cell type P3X as a production substrate
To the extent that 63Ag 8.653 Plasmacytoma cell line has been clarified, there is no mention of an industrially advantageous production method. Further, regarding the t-gD-IL-2 fusion protein which is expected to be developed as an anti-herpes simplex virus agent, CHO-HDL into which a gene of interest has been introduced has been introduced.
-1-5 cells or mouse myeloma Sp-neo-HD
A method using L-245 cells is disclosed in JP-A-4-117399.
However, it is described simply as "cultured in serum-free medium ASF104 medium (manufactured by Ajinomoto Co.)",
No specific culturing method is mentioned, and only culturing at the laboratory level was performed for the purpose of confirming expression, which is far from industrial production. In order to industrially mass-produce useful physiologically active peptides or proteins, the development of a serum-free medium that can be used satisfactorily for industrial substance production is an important issue. Therefore, the required serum-free medium conditions are (1) inexpensive, (2) the composition of the components is clear, there is no difference between lots, and moreover, (3) static culture is performed. Not only that, it can be used for long-term stirring culture and perfusion culture.

[0006]

The present inventors have found that animal cells such as t-gD-IL- which is an anti-herpesvirus agent.
2 Using a recombinant mouse myeloma cell or NT-3-producing CHO cell that produces a fusion protein, a method for mass-producing a desired physiologically active peptide or protein was examined, and as a result, the composition was inexpensive and the composition was low. We have succeeded in developing an industrial-grade serum-free medium that is clear and uniform with no difference between lots, and further studied to complete the present invention. That is, the present invention provides (1) a serum-free medium for animal cell culture, which comprises an inorganic or organic iron compound, cyclodextrin and a nonionic surfactant in a basal medium, (2) insulin, ethanolamine or the The medium according to the above 1, further comprising a derivative and selenious acid or a salt thereof, (3) the inorganic or organic iron compound is ferric chloride, ferrous chloride, ferrous sulfate, iron nitrate, citric acid The medium according to 1 or 2 above, which is one kind or a combination of 2 or more kinds of iron acid, iron oxalate, or iron lactate, and (4) above, wherein the nonionic surfactant is a pluronic surfactant. Or the medium described in 1 or 2 above, which further comprises at least one of (5) dexamethasone, protein hydrolyzate and amino acids, and (6) protein addition The degradation product is polypeptone, lactalbumin hydrolyzate, bactotryptone, and any one or a combination of two or more types of casamino acids, (7) ferric chloride, α-cyclodextrin, Pluronic surfactants, insulin,
The culture medium according to 1 or 2 above, which comprises ethanolamine, sodium selenite and polypeptone.
(8) The animal cell, wherein the animal cell is a mouse myeloma cell or a Chinese hamster ovary cell, (9) the animal cell using the medium (1, 2, 3, 4, 5, 6 or 7) A method for producing the physiologically active peptide or protein, characterized by culturing
(10) The production method according to 9 above, wherein the animal cell is a mouse myeloma cell or Chinese hamster ovary cell, (11) the production method according to 9 above, wherein the culture is suspension stirring culture, and (12) a bioactive peptide or protein. , The hormone, analgesic, growth factor, cytokine, enzyme, neurotransmitter, receptor, antibody or antigen for vaccine, (13) the growth factor belongs to the nerve growth factor (NGF) family The above is 1
2. The production method according to 2, wherein (14) the physiologically active peptide or protein is a hormone, an analgesic substance, a growth factor, a cytokine,
10. The production method according to 9 above, which is a fusion protein of the same species or different species selected from enzymes, neurotransmitters, receptors, antibodies or antigens for vaccines. (15) The fusion protein comprises an envelope glycoprotein of herpes simplex virus and interleukin-
15. The production method according to 14 above, which is a fusion protein consisting of 2.
(16) A mouse myeloma cell line Sp2 / 0-22-32-34 having t-gD-IL-2 producing ability.

The serum-free medium for culturing animal cells in the present invention is characterized by containing an inorganic iron compound or a low molecular weight organic iron compound, cyclodextrin and a nonionic surfactant. That is, the serum-free medium is a medium formed by adding each of the above-mentioned components to various basal mediums, and composed of substances whose contained components have been clarified chemically.

As the basal medium, a known serum-free medium capable of culturing animal cells by adding serum can be used. For example, Eagle MEM medium (H.
Eagle, Science, Volume 130, 432
Page (1959)), Dulbecco's modified Eagle medium (DM
EM) medium (R. Dulbecco and G. Freeman, Virology, Vol. 8, p. 396 (1959)),
Iscove's Medium (N. Iscove and F. Melchers, Journal Experimental Methods (J. Exp. Me
d.), 147, 923 (1978)), Ham F1
2 medium (RG Ham, Proceedings of National Academy of Science (Proc. Nat. A
cad. Sci.,), 53, 288 (1965)),
L-15 medium (A. Leibovitz, American Journal of Hygene, Vol. 78,
173 (1963)), RPMI 1640 medium (G.
E. Moore et al., The Journal of the American Medical Association (JAMA), No. 1.
99, p. 519 (1967)), E-RDF medium (Kyokuto Pharmaceutical), UC-102 medium (Nissui Pharmaceutical), UC-21.
2 medium (Nissui Pharmaceutical Co., Ltd.), Daigo T medium (Nippon Pharmaceutical Co., Ltd., JP-A-60-145088), TL-2 medium (Y. Shintani).
Et al., Applied Microbiology and Biotechnology (Appl. Microbiol. Biotechnol.), Vol. 27, p. 533 (1988)), and a medium in which these are mixed at an appropriate ratio. Above all, E-R
DF medium, Daigo T medium, TL-2 medium and the like are more preferably used.

The components that characterize the serum-free medium of the present invention will be described in detail below. In addition, the addition amount of each component shown in the text represents the addition mass of the component to be added to the basal medium powder for 1 L of the final liquid medium.
At the time of use, these are dissolved in water to make 1 L to prepare a serum-free medium for animal cell culture of the present invention. As the inorganic iron compound, ferric chloride, ferrous chloride,
Ferrous sulfate, iron nitrate and the like, and as the low molecular weight organic iron compound, a non-protein low molecular weight organic iron compound is preferable, and examples thereof include iron citrate, iron oxalate, and iron lactate. For the purpose of an iron donor, one kind or two or more kinds are used. In particular, inorganic iron compounds are preferable, and among them, ferric chloride and ferrous sulfate are more preferably used. For example, about 18 to 370 μmo as iron ions
1, preferably about 35 to 80 μmol. Specifically, for example, in the case of ferric chloride (6 hydrate), about 5
~ 100 mg, preferably about 10-20 mg may be added.

As the cyclodextrin, any of α-cyclodextrin, β-cyclodextrin or γ-cyclodextrin may be used. Above all, α
-Cyclodextrin is more preferably used. Further, a derivative obtained by chemically modifying the cyclodextrin such as methylation is also used. Specifically, for example,
In the case of α-cyclodextrin, about 1 to 5 g, preferably about 1.5 to 3 g may be added, and in the case of β-cyclodextrin, about 100 to 500 mg, preferably about 200.
~ 300 mg should be added. Any nonionic surfactant may be used as long as it does not show toxicity to cells, and preferred examples thereof include polyether nonionic surfactants. For example, propylene oxide (propylen
Pluronic surfactants that are copolymers of e oxide) and ethylene oxide, specifically, Pluronic F68, F88,
F77 and P65 (both manufactured by Asahi Denka Kogyo Co., Ltd.) and the like can be mentioned. Above all, Pluronic F68 is more preferably used. These nonionic surfactants are considered to act on the cell membrane of animal cells and change their permeability to reduce the effect of cell-inhibited product inhibition. Nonionic surfactants are generally
About 0.25 to 3 g, preferably about 0.5 to 2 g may be added.

In addition to the above components, it is also preferable to add insulin, ethanolamine or a derivative thereof, selenious acid or a salt thereof to the serum-free medium for culturing animal cells in the present invention. Examples of insulin include natural insulin, and insulin produced by a synthetic method or a gene recombination method, and a substance having an insulin action, for example, insulin-like growth factor (IGF-I).
Etc. are also included. Specifically, for example, in the case of insulin, about 0.5 to 20 mg, preferably about 1 to 5 mg may be added. In the case of IGF-I, about 0.01 to 0.1 mg, preferably about 0.02 to 0.05 mg may be added. Examples of ethanolamine or its derivative include ethanolamine, ethanolamine hydrochloride, phosphoethanolamine and the like. For example, about 8 to 80 μmol, preferably about 16 to 32 μmol may be added in terms of ethanolamine. Specifically, for example, ethanolamine may be added in an amount of about 0.5 to 5 mg, preferably about 1 to 2 mg. As selenious acid or a salt thereof, selenious acid,
Examples thereof include sodium selenite, potassium selenite, and barium selenite. For example, as selenium ion, about 5 × 10 ^{−3 to} 60 × 10 ⁻³ μmol, preferably about 1
It may be added to 5X10 ^-3 ~30X10 ^-3 μmol. Specifically, for example, in the case of sodium selenite, about 1 to 1
0 μg, preferably about 3 to 5 μg may be added.

The serum-free medium for animal cell culture of the present invention may be supplemented with at least one of dexamethasone, protein hydrolyzate and amino acid, in addition to the above components. About 0.5 for dexamethasone
4 mg, preferably about 1-2 mg should be added. Dexamethasone is more preferable, but alternatively, hormones such as hydrocortisone, dexamethasone, triiodothyronine, prostaglandin and the like can be added. Examples of the protein hydrolyzate include polypeptone, lactalbumin hydrolyzate, bactotryptone and casamino acid, and these can be used alone or in combination of two or more. Specifically, for example,
Polypeptone about 0.25-3 g, preferably about 0.5-1.5.
g should be added.

Any amino acid may be used as long as it is a constituent amino acid of a natural protein, and these are usually used by adding them at an appropriate ratio. Specifically, arginine, asparagine, aspartic acid, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine,
Examples thereof include amino acids such as glutamic acid and alanine. Although the addition amount varies depending on the basal medium used, for the purpose of reinforcing amino acids in the basal medium, for example, arginine about 100 to 800 mg, preferably about 400 mg, asparagine about 15 to 150 mg, preferably about 100 mg, aspartic acid about 10 to 10. 80 mg, preferably about 60 mg, cysteine, about 20-150 mg, preferably about 90 mg, glutamine, about 150-1000 mg, preferably about 600 mg, glycine, about 10-40 mg, preferably about 20 mg, histidine, about 20-
80 mg, preferably about 50 mg, isoleucine about 10 to 17
0 mg, preferably about 120 mg, leucine about 15 to 170 mg
Preferably about 110 mg, lysine about 40-200 mg, preferably about 150 mg, methionine about 5-60 mg, preferably about 30 mg, phenylalanine about 5-80 mg, preferably about 4
0 mg, proline about 20-80 mg, preferably about 40 mg, serine about 10-200 mg, preferably about 100 mg, threonine about 20-300 mg, preferably about 100 mg, tryptophan about 2-40 mg, preferably about 20 mg, tyrosine about 5-mg.
100 mg, preferably about 30 mg, valine about 10-150 mg
It is preferable to add about 100 mg.

In the serum-free medium for animal cell culture of the present invention, saccharides may be added in addition to the above components. Preferred sugars in this case include monosaccharides such as glucose, mannose, fructose and xylose, and disaccharides such as sucrose, and glucose and mannose are more preferably used. These sugars may be added to the initial medium and / or added during the culture. Serum-free medium for animal cell culture in the present invention, the components listed above are premixed and stored as a composition, and when used for culturing, they can be appropriately dissolved in water before use, and Each component may be dissolved in water and used for each culture. When the animal cells are transformed by introducing a foreign gene, the serum-free medium for animal cell culture in the present invention, in addition to the above components, depending on the selection marker used in the transformation, Agents such as methotrexate which is a selective agent, mycophenolic acid or G418 which is a neomycin derivative may be added.

The animal cells used in the present invention may be either free-floating cells or adherent cells.
Examples of floating cells include human Burkitt lymphoma (Namalwa) cells and mouse myeloma cells Sp2 /
Examples thereof include O-Ag14 and X63Ag8-653. Examples of the adherent cells include Chinese hamster ovary-derived CHO cells, baby hamster kidney-derived BHK cells, human cervical cancer-derived HeLa cells, mouse breast cancer-derived C-127 cells, and mouse fibroblast N cells.
Examples include IH / 3T3, BALB3T3, and Verots S3 derived from African green monkey kidney. In addition, cells that are originally adherent cells may also be prepared by suspension acclimation by a known method described in Japanese Patent Application No. 6-104221. Among them, mouse myeloma cells and CHO cells that have been acclimated to suspension are more preferably used. The physiologically active peptide or protein-producing animal cell used in the production method of the present invention may be a known strain having the above-mentioned properties. Further, it can be obtained by introducing a gene encoding the above-mentioned physiologically active peptide or protein into the above-mentioned animal cells by a known genetic engineering technique, if necessary. It is naturally advantageous to use a strain that highly expresses the desired physiologically active peptide or protein as the parent strain. As the animal cells producing the physiologically active peptide or protein used in the production method of the present invention, cells that can be subjected to suspension stirring culture are particularly preferably used.

Animal cells producing a physiologically active peptide or protein used in the production method of the present invention are cloned from a human genomic library or a human cDNA library to obtain human genomic DNA, mRNA or cDNA.
To obtain a gene encoding a target bioactive peptide or protein obtained by polymerase chain reaction (PCR) method or by chemical synthesis from
If necessary, a suitable promoter (for example, SV4
0-derived promoter, retrovirus promoter, cytomegalovirus promoter, etc.), promoter-prepro or promoter-signal (eg, IL-2 gene signal, etc.), and insert this into an appropriate vector to obtain It can also be obtained by transforming the above-mentioned animal cell with the obtained vector. As an example of the animal cell line thus obtained, specifically, for example, as a t-gD-IL-2 producing strain, mouse myeloma cell Sp-neo-HDL-245 strain (FERM BP-2810), and , Its high-producing strain Sp2 / 0-22-32-34 (IFO-50
442, NIBH P-14401), etc.
Examples of protein-producing strains belonging to the GF family include NG
As the F-producing strain, NGF used in Examples described later
-2 (NT-3) producing CHO-N2-1 strain and CHO
-N2-1 SF strain (FERM BP-4624) and the like.

The method for producing a physiologically active peptide or protein according to the present invention is characterized by using the above serum-free medium for culturing animal cells, and the desired physiologically active peptide or protein is produced by animal cells. There is no particular limitation as long as it can be done. Those that can be advantageously produced by the production method of the present invention include, for example, hormones,
Analgesics, growth factors, cytokines, enzymes, neurotransmitters, receptors, antibodies, antigens for vaccines, etc., and active derivatives or fusion proteins thereof are included.
Specifically, for example, as hormones, luteinizing hormone-releasing hormone (LH-RH), thyroid stimulating hormone-releasing hormone (TRH), insulin, somatostatin,
Growth hormone, prolactin, adrenocorticotropic hormone (ACTH), melanocyte stimulating hormone (MSH),
Thyroid stimulating hormone (TSH), luteinizing hormone (L
H), follicle-stimulating hormone (FSH), vasopressin, vasopressin derivative {desmopressin [Journal of the Endocrine Society of Japan, Vol. 54, No. 5, pp. 676-691 (1978)].
, Etc., oxytocin, calcitonin, parathyroid hormone (PTH), glucagon, gastrin, secretin,
Examples include pancreosymine, cholecystokinin, angiotensin, human placental lactogen, human chorionic gonadotropin (HCG), cerulein, motilin and the like.

Examples of the analgesic substances include enkephalin, enkephalin derivatives [see US Pat. No. 4,277,394, European Patent Application Publication No. 31567], endorphin, dinorphin, kyotorphin, and the like. As a growth factor, nerve growth factor (NGF)
Family (NGF, BDNF, NGF-2 (NT-3
, NT-4, NT-5, etc.), epidermal growth factor (EGF), fibroblast growth factor (FGF) family (aFGF, bFGF, INT-2, HST-).
1, FGF-5, FGF-6, etc.), somatomedin, T
Cell growth factors and the like can be mentioned. Examples of cytokines include interferon (α type, β type, γ type),
Interleukins (IL-1, -2, -3, -4,-
5, -6, -7, -8, -9, -10, -11, -1
2, -13, etc.), granulocyte colony stimulating factor (G-CS
F), granulocyte-macrophage colony stimulating factor (CM-
CSF), macrophage colony stimulating factor (M-CS
F), erythropoietin, thymopoietin, thymosin and the like. Examples of the enzyme include urokinase, tissue plasminogen activator, kallikrein and the like. As a neurotransmitter,
For example, bombesin, neurotensin, bradykinin, substance P and the like can be mentioned. Examples of the receptor include somatostatin receptor, LH-RH
(Luteinizing hormone releasing hormone) receptor, GH-R
H (growth hormone releasing hormone) receptor, CRH
(Adrenocorticotropin-releasing hormone) receptor, T
Examples include RH (thyroid stimulating hormone releasing hormone) receptors.

An antibody is a protein produced in vivo by an immune response as a result of antigen stimulation, and is an immunogen (antigen).
It has a specific binding activity with. That is, it specifically binds to various physiologically active substances such as proteins, polysaccharides, nucleic acids, and lipids, which are composed of substances showing antigenicity or aggregates thereof (for example, virus particles, pollen, indoor dust, etc.) Any active protein may be used.
Specifically, for example, mouse antibody, human antibody, mouse / human chimeric antibody, humanized antibody, single chain antibody,
Examples include single domain antibodies, bispecific antibodies and toxin fusion antibodies. In addition, for example, mouse / human chimera bispecific antibody, humanized bispecific antibody, single-chain bispecific antibody and the like obtained as a combination thereof can be mentioned. Specific examples thereof include anti-hepatitis B virus surface antigen monoclonal antibody, anti-tetanus toxin monoclonal antibody, anti-tumor associated antigen monoclonal antibody, and the like. Specific examples of the chimeric antibody include a mouse / human chimeric anti-human fibrin (hereinafter abbreviated as FIB) specific antibody, a mouse / human chimeric anti-human urokinase (hereinafter abbreviated as UK) antibody, a mouse / human Chimera anti-F
IB / anti-UK bispecific antibody [JP-A-5-76385] and the like. In particular, among the fragments constituting these antibodies, F (ab ′) ₂ , Fab ′, which retains the ability to bind to the antigen,
Fab, Fv, dAb fragments and the like are preferable as the antibody protein which can obtain the active form in high yield.

Examples of antigens (proteins or polypeptides) for vaccines include herpes simplex virus (HS)
V), varicella zoster virus (VZV), herpesvirus antigens such as cytomegalovirus (CMV), human immunodeficiency virus (HIV), and retrovirus including adult T-cell leukemia virus (HTLV-1) Virus antigens, hepadnavirus antigens including hepatitis B virus (HBS), non-A non-B hepatitis viruses (HCV, HEV), togavirus antigens including Japanese encephalitis virus, hepatitis A virus (HAV) and other picornavirus antigens, influenza virus and other orthomyxovirus antigens,
Parvovirus antigen, papovavirus antigen, adenovirus antigen, box virus antigen, reovirus antigen, paramyxovirus antigen, rapdovirus antigen, arenavirus antigen, coronavirus antigen, etc. Virus antigens, pathogenic protozoal antigens such as malaria antigens, and pathogenic bacteria such as B. pertussis antigens. As specific examples, herpes simplex virus (HSV) type 1 or type 2 surface antigen gD or gB, varicella zoster virus (VZV) surface antigen g
pI or gpIII, human immunodeficiency virus (HIV)
gag antigen or env antigen, adult T cell leukemia virus (HTLV-1) gag antigen or env antigen, C virus (HCV) C antigen, M antigen or E antigen, hepatitis B virus (HBV) C antigen ( HBc antigen), L protein, M protein or S protein (HBs).

The method for producing a physiologically active peptide or protein of the present invention can be preferably applied to the production of a physiologically active peptide or protein which is difficult to obtain in a large amount because it exists in a small amount in nature. Further, the production method of the present invention comprises a non-natural bioactive peptide or protein such as a natural bioactive peptide or protein or an active derivative thereof (mutein), and a fusion protein obtained by binding them. Can be used in any of the above, and its three-dimensional structure and the presence or absence of sugar chains are important factors for the expression of physiological activity, so that it is particularly advantageous for the production of physiologically active peptides or proteins that are preferably produced using animal cells. Can be used.

The fusion protein in the present invention is a genetically engineered combination of the same or different species selected from the above-mentioned physiologically active peptides or proteins. That is, hormones, analgesics, growth factors, cytokines, enzymes, neurotransmitters, receptors, antibodies, antigens for vaccines, etc., and those to which the same kind or different kinds selected from active derivatives or muteins thereof are bound Is. Specifically, for example, those in which an antigen for vaccine and cytokine are bound, those in which monoclonal antibody and growth factor are bound, those in which growth factor and cytokine are bound, those in which cytokines are bound, growth factor Among these, the present invention is preferably used for the production of a vaccine in which an antigen and a cytokine are bound. A specific example is the t-gD-IL-2 fusion protein. The production method of the present invention is also suitable for culturing a transformant produced to produce such a fusion protein.
Cloning methods for selection of high-producing clones are known, for example, colony separation method, microwell method (edited by the Japanese Biochemical Society, New Chemistry Laboratory 18, Cell Culture Technology, pages 12-13 (1990), Tokyo Kagaku Dojin). ) And the like.

Using the physiologically active peptide or protein producing strain obtained as described above, large-scale culture is carried out in a serum-free medium in order to produce a large amount of the desired physiologically active peptide or protein. As a culture apparatus at this time, a known floating agitation culture tank (Japanese agitation means, agitation means, temperature control means, pH control means, dissolved oxygen (DO) control means and the like necessary for culture is provided. Biochemical Society
Newborn Chemistry Laboratory 1, Protein VI Synthesis and Expression No. 2
82, 286 (1992), Tokyo Kagaku Dojin; Japan Biochemical Society, Shinsei Chemistry Laboratory, 18, Cell Culture Technology, 31.
Pages 3-323 (1990), Tokyo Kagaku Dojin, etc. are used. In addition, an air-lift culture tank (JR Birch et al., Trends in Biotec
hnol.) Vol. 3, p. 162 (1985).
Examples of the culture method include batch culture, feed culture (fed / batch culture), and perfusion culture. In the feed culture, the concentrated liquid of the medium and / or the concentrated liquid of sugar, more preferably only the sugar liquid, is fed into the culture liquid once during the culture or continuously or intermittently during the culture to continuously or intermittently feed the culture liquid. It is possible to prevent nutrient depletion and maintain production while keeping cells alive. Glucose or mannose is used as the sugar to be fed. In order to carry out the feed culture, a feed liquid storage tank and a feed liquid supply means are provided in connection with the culture tank. The feed is usually started at the time when the bacterial cells reach the logarithmic growth phase, usually immediately after the start of culture to 6th day, preferably about 3 days.
From day one. In batch culture, a method of adding 1 to 10 g / L, preferably 3 to 5 g / L of sugar at the time of medium preparation is adopted. In the perfusion culture, the culture tank is provided with a means for separating cells in the culture medium from the culture supernatant, a means for discharging the culture supernatant, and a means for supplying fresh medium. As a means for separating the cells in the culture medium from the culture supernatant liquid, for example, a known separation method using a filter, a separation method utilizing the sedimentation of cells such as a cone-shaped cell settling tube or a gravity settling tube, or a centrifugation method is known. Separation means may be mentioned. For example, a method in which a culture system in which these separating means are provided in a culture tank is used and a culture supernatant is continuously discharged and replaced with a fresh medium by a known method is preferably used. The discharge of the culture supernatant and the supply of the fresh medium, that is, the medium exchange, are usually performed from the logarithmic growth phase to the stationary phase, usually about 2 to 10 days after the start of the culture, preferably about 3 to 7 days. It is preferable that the exchange rate of the medium is gradually increased as the cell density is increased. Specifically, for example, about 10 to 2 of the culture solution volume per day of culture is used.
It is preferred to replace it by 00%, especially about 50-150%.

The temperature during culturing is usually about 30 to 40 ° C., preferably around 37 ° C., the stirring speed is about 20 to 100 rpm,
Preferably around 30 rpm, pH about 6-8, preferably 7
The DO is adjusted to about 0.5 to 5 ppm, preferably to about 1.5 ppm. The target physiologically active peptide or protein is collected from the culture solution obtained by withdrawing it during the culture, or from the culture solution recovered after the completion of the culture.

In order to separate and purify the physiologically active peptide or protein produced or accumulated intracellularly or extracellularly by the production method of the present invention, known separation and purification methods may be appropriately combined. Known separation and purification methods thereof include a method utilizing a difference in solubility such as salting out, ammonium sulfate precipitation and solvent precipitation, dialysis, ultrafiltration, and S.
A method mainly utilizing a difference in molecular weight such as DS-polyacrylamide gel electrophoresis, a method utilizing a difference in charge such as ion exchange chromatography, a method utilizing a specific compatibility such as affinity chromatography, for example, an antibody Columns and metal chelate columns such as Cu ²⁺ columns, reverse phase high performance liquid chromatography (H
Examples thereof include a method of utilizing a difference in hydrophobicity such as PLC) and a method of utilizing a difference in isoelectric point such as isoelectric focusing.

[0026]

The present invention will be described in detail below with reference to examples.
The present invention is not limited to this. The t-gD-IL-2 producing mouse myeloma cell Sp-neo-HDL-245 strain used in the examples described below was deposited under the Accession No. FERM at the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (NIBH), Ministry of International Trade and Industry. Deposited as BP-2810. The t-gD-IL-2 high-producing clone Sp2 / O-22-32-34 strain obtained in Example 12 was
From the 13th of April, 2013, the Fermentation Research Institute (IFO) with the accession number IFO 50442, and June 29, 1994.
It has been deposited at the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (NIBH) with the deposit number FERM P-14401 from the day.

Example 1 L-containing E-RDF medium powder (Kyokuto Pharmaceutical Co., Ltd.), ferric chloride (6 hydrate) 10 mg, α-
A medium was prepared by adding 2.2 g of cyclodextrin and 1 g of Pluronic F68, and further adding 10 mg of insulin, 1.5 mg of ethanolamine, 4 μg of sodium selenite, 1 mg of dexamethasone and 1 g of polypeptone, and dissolving this in water to prepare 1 L. Of serum-free medium. Sp-neo-H
One cryopreserved vial of the DL-245 strain was taken out from the liquid nitrogen storage container, rapidly thawed at 37 ° C, mixed with about 9 ml of the above serum-free medium and centrifuged. The centrifugation supernatant was discarded, and the remaining cells were resuspended in about 50 ml of the above serum-free medium (the number of viable cells was about 1 × 10 ⁵ cells / ml) and placed in an F75 flask in a 2.5% carbon dioxide gas incubator. At 37 °
C, cultured overnight. The next day, the medium was replaced with the fresh serum-free medium described above, and the cells were cultured at 37 ° C for 2 days. The cells that have grown are collected by centrifugation and placed in a 125 ml Erlenmeyer flask into which 50 ml of the above serum-free medium has been dispensed.
It was implanted in such a way that 0 ⁵ cells / ml. Glucose was fed at 3 g / L on the 3rd day of the culture while the pH was adjusted to near neutral in a 37 ° C. rotary shaker, and the cells were cultured for 8 days. In the serum-free medium, ferric chloride (6-hydrate) -free medium was used, and the same culture was performed as a comparative experiment. As a result, good growth as ferric added chloride, cultured for 3 days in 9 × 10 ⁵ cells / ml, 25 × 10 ⁵ cells on day 5 culture /
Although the number of cells reached to ml, the cells without addition had extremely poor growth and reached 2 × 10 ⁵ cells / ml on the 3rd day of culture, and the cells died thereafter. As shown in the production amount of t-gD-IL-2 in [Table 1], it was found that the growth and the productivity are increased by the addition of ferric chloride, which is an inexpensive inorganic iron compound.

[0028]

[Table 1] Embodiment 2. FIG. Effect of addition of insulin t-gD-IL-2 producing Sp-neo-HDL-245 cells were added to a 125 ml Erlenmeyer flask into which 50 ml of serum-free medium was dispensed at 0.8 × 10 ⁵ cells / ml. I planted it. In this experiment, ferric chloride (6 hydrate) 10 mg, ethanolamine 1.5 mg, sodium selenite 4 μg, α were added to 1 L of E-RDF medium powder (Kyokuto Pharmaceutical Co., Ltd.).
-2.2 g of cyclodextrin, 1 g of Pluronic F68,
A medium containing 1 mg of dexamethasone and 1 g of polypeptone was prepared and dissolved in water to give 1 L of serum-free medium. Insulin or insulin-like growth factor I
(IGF-I) added at the concentration shown in [Table 2],
Alternatively, the additive-free one was used. Glucose was fed at 3 g / L on the 3rd day of culture while the pH was adjusted to near neutral in a 37 ° C. rotary shaker, and the cells were cultured for 8 days. Insulin 1 mg / L or more was added and IGF-I. 25 μg / L
The added one grew better, the growth rate was faster and the maximum density reached was higher than that of the non-added one. t-gD-
As shown in [Table 2], the amount of IL-2 produced was almost the same for the one with 1 mg / L insulin (hereinafter referred to as E-RDF-4 medium) and the one with IGF-I 25 μg / L addition. Met. That is, when the medium having the above composition is used, insulin is added in an amount of about 1/5 to 1/10 of the amount generally added.
It turns out that the amount is good.

[0029]

[Table 2] Example 3. Production of t-gD-IL-2 by stirring large-scale culture t-gD-IL-2 producing Sp-neo-HDL-245 cells was dispensed with about 100 ml of E-RDF-4 serum-free medium, and the volume was 125 ml. About 1 viable cells in the spinner flask
The cells were transplanted at 10 ⁵ / ml and cultured at 37 ° C for 3 days with stirring. Add 500 ml of techne
The spinner flask was sequentially transferred to a 3-liter techne-spinner flask and cultured with stirring. The total volume of the culture solution (about 3
L), 50 L containing about 17 L of E-RDF-4 medium
The cells were transferred to a stirring culture tank, and cultivation was started at 37 ° C. and a stirring rotation speed of 30 rpm. On the 3rd day of culture, glucose was fed in an amount of 3 g / L, the pH was controlled to around 7.0, and the dissolved oxygen was controlled to around 1.5 ppm, and the cells were cultured for 7 days, resulting in about 24 mg / L.
t-gD-IL-2 was produced. The progress of culture at this time is shown in FIG. As a result, the serum-free medium of the present invention is
It was found that pre-culture and main culture can be used for large-scale culture of animal cells.

Example 4. Isolation and purification of t-gD-IL-2 19 L of the culture broth obtained in Example 3 was centrifuged to obtain 18.4 L of culture supernatant. Add 2 ammonium sulfates to this
20% tris-containing 20% saturated ammonium sulphate and 0.1 mM (p-amidinophenyl) methanesulfonyl fluoride after adding 0% saturation.
It was added to a butyl-Toyopearl 650M column (10 × 4 cm) equilibrated with a hydrochloric acid buffer (pH 7.5), washed with the same buffer, and then washed with 5% saturated ammonium sulfate and 0.1 mM (p-amidinophenyl) methanesulfonylfluoride. Elute with 20 mM Tris-hydrochloric acid buffer solution (pH 7.5) containing ride, and
Fractions of 2.1 L containing gD-IL-2 were collected. This eluate fraction was added to a Trisacryl GF05 column (15 × 70 cm) equilibrated with a 20 mM phosphate buffer solution (pH 6.3) containing 0.1 mM (p-amidinophenyl) methanesulfonyl fluoride in advance, and the same fraction was added. Elute with buffer
2.4 L of the fraction containing t-gD-IL-2 was collected. This elution fraction was preliminarily set to 0.1 mM (p-amidinophenyl)
After adding to a Super Q-Toyopearl 650M column (5 x 15 cm) equilibrated with a 20 mM phosphate buffer solution (pH 6.3) containing methanesulfonyl fluoride and washing with the same buffer solution, sodium chloride concentration in the same buffer solution was added. 0 to 400
Elute with a linear gradient elution method of increasing to mM, t-gD-I
210 ml of the main elution fraction of L-2 was collected. 35 ml of the concentrated solution obtained by concentrating this fraction in a diaflow cell (PM-10 membrane, manufactured by Amicon) was preliminarily used in 25 mM ammonium acetate buffer (pH: 150 mM sodium chloride).
6.5) was loaded onto a Sephacryl S-300HR column (5 x 90 cm) equilibrated and eluted with the same buffer to obtain t-gD-
120 ml (52 mg) of purified IL-2 preparation was obtained.

SDS-polyacrylamide gel electrophoresis (gel for separation: 10-20% gradient gel; reducing condition: 10% 2-mercaptoethanol, 100 ° C., 5 minutes; staining: Coomassie Brilliant Blue) using this purified sample As a result of performing R250), a single band was obtained as shown in FIG. The results of amino acid composition analysis of this purified preparation are shown in [Table 3], the results of N-terminal amino acid sequence analysis are shown in [Table 4], and the results of C-terminal amino acid analysis are shown in [Table 5]. As a result of these protein chemical analyzes, the purified sample obtained here was in good agreement with that expected from the nucleotide sequence.

[0032]

[Table 3]

[0033]

[Table 4]

[0034]

[Table 5]

Example 5. T-gD-I by cloning
Selection of L-2 high-producing clonal strain For the purpose of selecting a high-producing clonal strain from the Sp-neo-HDL-245 strain, cloning by the limiting dilution culture method was performed.
Repeated times. That is, the culture solution was collected from the culture flask, centrifuged, and the cells were collected and suspended in a cloning medium (1% FCS (fetal calf serum) and 4 mg / L neomycin derivative G418 added to ASF-104 medium). , And diluted with the same medium so that the number of cells was 5 per ml. 0.1 ml of this solution was dispensed into each well of about 15 96-well plates, and cultured in a 2.5% carbon dioxide gas incubator at 37 ° C. for 15 days. Wells in which cell growth was observed were selected, and 0.1 ml of fresh cloning medium was added. After culturing for another 5 days, 0.5 ml
ASF-104 serum-free medium (G418 4mg / L added)
Was transplanted to a 24-well plate in which the above was dispensed. The transplantation amount was 0.1 ml as a standard, and was appropriately increased or decreased according to the growth situation. 37 ° C in a 2.5% carbon dioxide incubator, 3
After culturing for one day, 100 μl of the culture solution in each well was collected and subcultured to a new 24-well plate. After culturing the rest for 2 more days, the amount of t-gD-IL-2 produced was measured by EIA. Twenty-four clones showing relatively high productivity were selected, and cells were transferred from the plate subcultured on the third day into 12-well plates, F25 flasks, F75 flasks and 150 flasks, and E-RDF-4 medium (G4
18400 μg / L was added) and the cells were successively passaged and cultured. Furthermore, these 24 clones were transplanted into a 125 ml techne spinner flask at 1 × 10 ⁵ / ml to prepare E-RDF-4.
The clones with the highest productivity were selected by culturing with stirring in a medium (G418 400 μg / L added).

Next, in order to select a clone with higher production, cloning and selection were carried out in the same manner, and finally a stable clone with high production Sp2 / O-22-32-34 was selected. Sp2 / O-22 obtained in this way
Strain 32-34 was cultured in a shake flask, and the productivity was compared. That is, the cells were placed in a 125 ml Erlenmeyer flask into which 50 ml of serum-free medium was dispensed, and 0.8 x
The cells were seeded at 10 ⁵ cells / ml. 1 L of Daigo T is used for serum-free medium for evaluating the productivity of high-producing clonal strains.
Amino acid (arginine 400) in medium powder (Nippon Pharmaceutical)
mg, asparagine 100 mg, aspartic acid 60 mg, cysteine 90 mg, glutamine 600 mg, glycine 20 m
g, histidine 50 mg, isoleucine 120 mg, leucine 110 mg, lysine 150 mg, methionine 30 mg, phenylalanine 40 mg, proline 40 mg, serine 100 m
g, threonine 100 mg, tryptophan 20 mg, tyrosine 30 mg, valine 100 mg), insulin 1 mg, ferric chloride (6 hydrate) 10 mg, ethanolamine 1.5 m
g, sodium selenite 4 μg, α-cyclodextrin 2.2 g, Pluronic F68 1 g, dexamethasone 1 m
A medium containing 1 g of polypeptone and 1 g of polypeptone was prepared and dissolved in water to give 1 L of a serum-free medium (hereinafter referred to as ET-4 medium). 4 g of glucose was added to this medium. When cultured for 8 days in a 37 ° C. rotary shaker while adjusting the pH to near neutral, Sp2 / O-22-
For strain 32-34, t-gD- of about 102 mg / L (3 times average)
IL-2 was produced. For comparison, when the original strain Sp-neo-HDL-245 was similarly cultured, t
-GD-IL-2 productivity is about 24 mg / L (3 times average)
Met. Therefore, a high-producing clonal strain in which the productivity of t-gD-IL-2 was increased by about 2.4 times compared with the original strain was obtained.

Example 6. Human N by culturing CHO cells
Production of T-3 Human NT-3-producing CHO-N2-1 strain (Iwane et al., JP-A-5-103675), serum-free suspension agitated CHO-N strain
2-1 SF (FERM BP-4624) was placed in a 125 ml Erlenmeyer flask in which 50 ml of each of 5 types of serum-free medium (see the description in the table) was dispensed, and the number of viable cells was about 1 × 10 ⁵ / Transplanted to be ml. 37 ° C rotary shaker
In (100 rpm), adjust the pH to around neutrality 16
After culturing for a day, the amount of NT-3 in the supernatant of the culture broth was measured with enzyme immunoassay (EIA) [Shinya et al., Biochemical and Biophysical Research Communication (BBRC), 194, 1500 (1993). ))
When measured by, the results shown in [Table 6] were obtained. That is, ASF-104 medium or COSMEDIUM-0 which is widely used as an excellent medium.
It was proved that the E-RDF-4 medium and ET-4 medium of the present invention can be advantageously used in human NT-3-producing CHO cells as compared with 01 medium and the like.

[0038]

[Table 6]

Example 7. T-gD-IL-by perfusion culture
Mass production of 2 The Sp2 / O-22-32-34 strain obtained in Example 4 was added to about 100 ml of ET-4 medium (glucose 4 g / l added).
Were transplanted into a 125 ml techne-spinner flask into which the viable cells were dispensed so that the number of viable cells would be approximately 1 × 10 ⁵ / ml, and the temperature was maintained at 37 ° C.
The culture was carried out with stirring for one day. The proliferated cells are further added to a 500 ml techne spinner flask, and the number of viable cells is about 1 × 10 ⁵ / m 2.
The cells were transplanted so as to have a volume of 1 and cultured at 37 ° C. until the latter phase of the logarithmic growth phase. The grown cells were transplanted at a seeding density of about 1 × 10 ⁵ / ml into a 2 L jar fermenter charged with about 1 L of ET-4 medium (containing 4 g / l glucose),
Culturing was started at 37 ° C. and a rotation speed of 40 rpm. From the 4th day of culture, perfusion was started using ET-4 medium (containing 4 g / l of glucose) at a medium exchange rate of about 70% (medium exchange rate per day of culture per 1 L of culture solution), and cells were cultured. The medium exchange rate was gradually increased to about 150% with increasing density. PH of the culture solution is around 7.0, dissolved oxygen (DO)
Was controlled to about 1.5 ppm and cultured. as a result,
After culturing for 35 days, the total number of cells grew to a density of about 4 × 10 ⁷ / ml, and an integrated amount of about 1500 mg of t-gD-IL-2 was produced.

[0040]

EFFECTS OF THE INVENTION The serum-free medium for culturing animal cells of the present invention is (1) inexpensive, (2) the composition of the components is clear, and there is no difference between lots, and () 3) It can be used for culturing animal cells through pre-culture and main culture without adding serum, and (4) not only static culture but also stirring culture (batch culture, fed-batch culture, etc.) or long-term It can be used extremely advantageously for industrial production of various physiologically active peptides or proteins, such as the fact that perfusion culture is possible. One of the characteristics of the serum-free medium is that it does not use an iron donor derived from serum and uses an inorganic or non-protein organic low-molecular weight organic iron compound, which makes it possible to inexpensively supply the medium, Moreover, the productivity is also good. By industrial production by animal cell culture using the serum-free medium, specifically, for example, NGF, those belonging to the NGF family such as NT-3, which are expected to be applied to senile dementia, immunity against herpes simplex virus. Fusion protein t-gD- whose development is expected as a highly original vaccine
Large-scale supply of IL-2 etc. becomes possible.

[Brief description of drawings]

FIG. 1 t-gD-IL-2 producing Sp-neo according to Example 3.
-Progress of large-scale culture of HDL-245 cells is shown.

FIG. 2 shows the results of SDS-polyacrylamide gel electrophoresis of the purified t-gD-IL-2 preparation obtained in Example 4.

[Explanation of symbols]

M is a molecular weight marker, A is t-gD obtained in Example 4.
-Shows a purified preparation of IL-2.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI technical display location C12N 5/02 7729-4B C12P 21/02 K 9282-4B H 9282-4B // (C12N 5 / 10 C12R 1:91) (C12P 21/02 C12R 1:91) (C12N 5/00 B C12R 1:91)

Claims (16)

[Claims] 1. A serum-free medium for animal cell culture, which comprises an inorganic or organic iron compound, cyclodextrin and a nonionic surfactant in a basal medium. 2. The medium according to claim 1, further comprising insulin, ethanolamine or a derivative thereof, and selenious acid or a salt thereof. 3. An inorganic or organic iron compound is ferric chloride,
The medium according to claim 1 or 2, which is any one of ferrous chloride, ferrous sulfate, iron nitrate, iron citrate, iron oxalate, or iron lactate, or a combination of two or more thereof. 4. The medium according to claim 1 or 2, wherein the nonionic surfactant is a pluronic surfactant. 5. The medium according to claim 1, which is supplemented with at least one of dexamethasone, protein hydrolyzate and amino acids. 6. The medium according to claim 5, wherein the protein hydrolyzate is any one of polypeptone, lactalbumin hydrolyzate, bactotryptone and casamino acid, or a combination of two or more thereof. 7. The medium according to claim 1 or 2, which contains ferric chloride, α-cyclodextrin, a pluronic surfactant, insulin, ethanolamine, sodium selenite and polypeptone. 8. The medium according to claim 1, wherein the animal cell is a mouse myeloma cell or a Chinese hamster ovary cell. 9. An animal cell is cultivated using the medium according to claim 1, 2, 3, 4, 5, 6 or 7 to produce and accumulate a physiologically active peptide or protein, which is then collected. A method for producing the physiologically active peptide or protein. 10. The method according to claim 9, wherein the animal cell is a mouse myeloma cell or a Chinese hamster ovary cell. 11. The production method according to claim 9, wherein the culture is suspension stirring culture. 12. The method according to claim 9, wherein the physiologically active peptide or protein is a hormone, analgesic substance, growth factor, cytokine, enzyme, neurotransmitter, receptor, antibody or antigen for vaccine. 13. The method according to claim 12, wherein the growth factor belongs to the nerve growth factor (NGF) family. 14. The bioactive peptide or protein is a fusion protein of the same species or different species selected from hormones, analgesics, growth factors, cytokines, enzymes, neurotransmitters, receptors, antibodies or antigens for vaccines. Item 9. The production method according to Item 9. 15. The method according to claim 14, wherein the fusion protein is a fusion protein consisting of an envelope glycoprotein of herpes simplex virus and interleukin-2. 16. A mouse myeloma cell line Sp2 / 0-22-32-34 capable of producing t-gD-IL-2.