JP3822137B2 - Additive for animal cell culture medium and method for producing protein using the same - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an additive for animal cell culture medium, a method for producing the additive, an animal cell culture medium containing the additive, and a protein production method using the same. Specifically, the present invention includes an enzyme degradation product of a mixture of fish meat viscera and true meat or an extract of the mixture, and an animal cell containing the additive. The present invention relates to a culture medium and a protein production method using the same.
[0002]
[Prior art]
When culturing animal cells to obtain a natural protein produced by the animal cells, or when culturing animal cells introduced with a gene encoding a desired protein to produce a desired protein, In addition to basic nutrients such as salts, saccharides, amino acids, and vitamins, extracts from mammals, specifically serum such as fetal calf serum, are usually used for the growth of the animal cells. % Is added to the medium. However, such mammal-derived serum has the disadvantage that it accounts for 75 to 95% of the cost of the culture medium and that stable growth cannot be obtained due to the difference in lot quality. In addition, since mammal-derived serum cannot be sterilized by autoclaving or the like, it may be contaminated with virus or mycoplasma, many of which are harmless, but may be an additional unknown factor in terms of stable production. Furthermore, serum contains more than 500 kinds of proteins, which complicates the isolation and purification of desired proteins, which are cell products from the culture medium. In order to solve such a problem in stable production, a method of using a purified protein derived from serum such as fetuin, insulin, transferrin or the like instead of serum has been performed. In addition, from the viewpoint of production cost, an attempt has been made to use a medium component extracted from a mammal.
[0003]
In recent years, however, the components derived from mammals are mad cow disease, bovine spongiform encephalopathy (BSE), infectious spongiform encephalopathy (Transmissible Spongiform Encephalopathy: TSE), and even Creutzfeldt. -Correlation with Creutzfeld-Jakob Disease (CJD) and the like is concerned, and from the viewpoint of safety, the emergence of animal cell culture media that do not contain these mammal-derived components has been desired.
[0004]
However, when cultivating animal cells, if the above-mentioned components derived from mammals are not added to the medium to be used, the cell viability is significantly reduced at an early stage of culture, and the number of viable cells in the culture solution Therefore, there is a problem that long-term culture or mass culture cannot be performed.
[0005]
In International Publication No. WO99 / 63058, it is found that long-term culture of animal cells and large-scale culture of desired proteins are possible by adding fish meat including fish meat and viscera instead of mammal-derived components. ing. However, WO99 / 63058 does not mention what kind of influence is exerted on the amount of protein produced when the ratio of viscera and true meat in fish meat is changed.
[0006]
[Problems to be Solved by the Invention]
An object of the present invention is to provide a medium that is particularly effective among animal cell culture media containing fish extract or fish enzyme degradation product.
[0007]
[Means for Solving the Problems]
As a result of intensive efforts to achieve the above-mentioned problems, the inventors of the present invention are composed of fish internal organs and meat compared to animal cell culture media containing only fish internal organs or only meat. It has been found that an animal cell culture medium to which an enzyme degradation product of the mixture or an extract of the mixture has been added is particularly effective, and the present invention has been completed. In addition, the inventors of the present invention have a cell viability and protein production when the weight ratio of viscera to meat is 75:25 to 40:60, preferably 65:35 to 55:45, and more preferably 60:40. The amount was found to improve.
[0008]
That is, the present invention provides an additive to an animal cell culture medium, comprising an enzymatic degradation product of a mixture of fish meat viscera and meat, or an extract of the mixture.
[0009]
The present invention further provides a method for producing an additive in an animal cell culture medium comprising the following steps:
(A) a step of mixing the internal organs and the true meat of fish meat, and (b) a step of enzymatically decomposing the mixture to obtain an enzymatic degradation product, or obtaining an extract from the mixture.
[0010]
The present invention further provides a method for producing an additive in an animal cell culture medium comprising the following steps:
a) Enzymatic degradation of fish internal organs to obtain an enzymatic degradation product, or obtaining an extract from the internal organs,
(B) Enzymatic degradation of fish meat to obtain an enzymatic degradation product, or obtaining an extract from the true meat, and (c) the substance produced in the step (a) and the step (b) The process of mixing the produced substance.
[0011]
The present invention further provides an animal cell culture medium containing the additive.
The present invention further includes a method for producing a desired protein by culturing animal cells, wherein the culture includes an enzymatic degradation product of a mixture of fish internal organs and meat or an extract of the mixture. Provided is a method for producing a protein using a cell culture medium.
[0012]
The present invention further provides a protein produced by the above method.
[0013]
DETAILED DESCRIPTION OF THE INVENTION
In the additive for animal cell culture medium of the present invention, the weight ratio of viscera to meat is 75:25 to 40:60, preferably 65:35 to 55:45, and more preferably 60:40.
[0014]
When a medium containing the additive of the present invention is used, it is possible to cultivate animal cells particularly well without adding a mammal-derived component. In addition, a good protein production amount can be obtained.
[0015]
Fish for collecting the internal organs and true meat of the meat used in the present invention include red fish such as salmon, soda bonito, salmon, salmon, sword fish, salmon, salmon, salmon, and white meat such as salmon, salmon, flat eyes, salmon, salmon Examples include fish, preferably salmon, soda bonito, salmon, salmon, salmon, salmon, and particularly preferably salmon and salmon.
[0016]
The meat and viscera to be mixed may be the same fish species, but the fish and viscera may be different fish species.
As the additive used in the present invention, an enzymatic degradation product of a mixture of fish meat viscera and true meat or an extract of the mixture can be used. The enzyme degradation product of the mixture can be obtained by treating the mixture or mixture extract with a proteolytic enzyme.
[0017]
Alternatively, the internal organs of fish meat can be enzymatically decomposed to obtain an enzymatic decomposition product, or an extract can be obtained from the internal organs, and the fish meat can be enzymatically decomposed to obtain an enzymatic decomposition product, or an extract from the true meat can be obtained. After obtaining, an additive may be manufactured by mixing these.
[0018]
The extract of the fish meat mixture used in the present invention includes, for example, the above-mentioned fish meat viscera and the true meat cut together or separately into appropriate pieces, or minced into a paste form, and the soluble component is heated with hot water. For example, it can be obtained by extraction with hot water of 90 to 95 ° C. for several tens of minutes to several tens of hours. Specific examples include boiled simmered broth at the time of bonito manufacture and cook drain at the time of canned manufacture.
[0019]
In addition, the enzymatic decomposition product of the fish meat mixture was obtained by, for example, cooking the fish internal organs and the true meat together or separately, or as a paste made from mince, or obtained as described above. It can be obtained by adding an appropriate amount of water to the extract, heating it as necessary to denature the protein, treating it with a proteolytic enzyme, and removing oils and insolubilized materials by centrifugation or filtration as appropriate. It is preferable to use the extract or enzymatic degradation product of such a fish meat mixture after adjusting the pH to about 7-7.4.
[0020]
Examples of proteolytic enzymes used in the present invention include proteinases and / or peptidases. In the present invention, the term proteinase refers to an enzyme that hydrolyzes a protein using the protein as a substrate, and the term peptidase refers to a peptide-bonded hydrolase that uses the peptide as a substrate. That is, the activity of a protease on a protein substrate can be distinguished as proteinase activity, and the activity on a peptide substrate can be distinguished as peptidase activity. The term proteinase is used to catalyze the cleavage of the peptide bond chain from the middle by the activity of a protease on a protein substrate, and thus endopeptidase is used herein as a type of proteinase. The proteinase or peptidase used in the present invention is preferably not derived from a mammal.
[0021]
Specifically, enzymes of plant origin such as papain, chymopapain, promelain, ficin and enzymes of microorganism origin such as mold, bacteria, yeast, etc., enzymes such as endopeptidase, exopeptidase, aminopeptidase, carboxypeptidase, dipeptidase Etc. These enzymes can be used alone or in combination. When used in combination, they may be added simultaneously or stepwise.
[0022]
The enzymatic decomposition product of the fish meat mixture in the present invention is preferably an enzymatic decomposition product of the fish meat mixture obtained by treating with the above proteinase and then treating with the peptidase.
[0023]
The enzyme treatment varies depending on the type of enzyme used, but is usually pH 2-12, preferably pH 4-8, 30-90 ° C, preferably 40-65 ° C, 30 minutes-72 hours, preferably 3 -Perform for 24 hours. At that time, the enzyme is used in an amount of 0.01-10%, preferably 0.5-5%, more preferably 1-3% of the protein as a substrate.
[0024]
After deactivating the enzyme in the enzymatic decomposition product of the fish meat mixture obtained in this way by heating, etc., the enzymatic decomposition product is prepared by removing the oil and insolubilized material by appropriate centrifugation, filtration, etc. can do.
[0025]
The obtained enzyme degradation product can be concentrated as necessary, or can be powdered by using drying and powdering techniques such as spray drying and freeze drying, and can be used as an additive to a culture medium for animal culture.
[0026]
Examples of the internal organs of fish meat in the present invention include stomach, pylorus, intestine, liver, gallbladder, testis, ovary, heart and the like. The true meat of the fish meat in the present invention refers to a real meat portion obtained by removing the above-mentioned internal organs, bones, heads and the like from fish.
[0027]
The additive to the animal cell culture medium of the present invention is not particularly limited as long as it contains fish viscera and true meat, but is not particularly limited in terms of cell viability and protein production. Has a weight ratio of viscera to true meat of 75:25 to 40:60, preferably 65:35 to 55:45, and more preferably 60:40.
[0028]
In addition, in this invention, when manufacturing the mixture which consists of the internal organs and true meat of a fish meat, parts, such as a fish bone and a head, may mix.
The animal cell culture medium of the present invention contains an additive to the animal cell culture medium characterized by containing the enzymatic degradation product of the mixture of the fish viscera and the meat of the present invention or the extract of the mixture. In addition, other components that are usually used in animal cell culture media can be appropriately used as other components. These include amino acids, vitamins, lipid factors, energy sources, osmotic pressure regulators, iron sources, pH buffers. In addition to the above components, for example, trace metal elements, surfactants, growth cofactors, nucleosides and the like may be added.
[0029]
Specifically, for example, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-cystine, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-ornithine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, etc., preferably L-alanine , L-arginine, L-asparagine, L-aspartic acid, L-cystine, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L- Amino acids such as phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine; i-inositol, biotin, folic acid, lipoic acid, nicotinamide, nicotinic acid, p- Aminobenzoic acid, calcium pantothenate, pyridoxal hydrochloride, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, vitamin B12, ascorbic acid, etc., preferably biotin, folic acid, lipoic acid, nicotinamide, calcium pantothenate, pyridoxal hydrochloride, riboflavin, thiamine hydrochloride Vitamins such as vitamin B12 and ascorbic acid; choline chloride, choline tartrate, linoleic acid, oleic acid, cholesterol, etc., preferably lipid factors such as choline chloride; energy such as glucose, galactose, mannose, fructose, preferably glucose Source: Sodium chloride, potassium chloride, potassium nitrate, etc., preferably osmotic pressure regulator such as sodium chloride; EDTA iron, iron citrate, ferrous chloride, ferric chloride, ferrous sulfate, ferric sulfate, nitric acid Ferric iron, etc. Preferably iron sources such as ferric chloride, EDTA iron, iron citrate; sodium bicarbonate, calcium chloride, sodium dihydrogen phosphate, HEPES, MOPS, etc., preferably including pH buffer such as sodium bicarbonate A culture medium can be illustrated.
[0030]
In addition to the above components, for example, trace metal elements such as copper sulfate, manganese sulfate, zinc sulfate, magnesium sulfate, nickel chloride, tin chloride, magnesium chloride, sodium silicate and the like, preferably copper sulfate, zinc sulfate, magnesium sulfate and the like; Surfactants such as Tween80 and Pluronic F68; and recombinant insulin, recombinant IGF, recombinant EGF, recombinant FGF, recombinant PDGF, recombinant TGF-α, ethanolamine hydrochloride, selenite Sodium, retinoic acid, putrescine hydrochloride, etc., preferably sodium selenite, ethanolamine hydrochloride, recombinant IGF, putrescine hydrochloride and other growth cofactors; deoxyadenosine, deoxycytidine, deoxyguanosine, adenosine, cytidine, guanosine, uridine, etc. The nucleoside may be added. The preferred embodiment of the present invention may contain antibiotics such as streptomycin, penicillin G potassium and gentamicin, and a pH indicator such as phenol red.
[0031]
In order to specifically prepare the animal cell culture medium of the present invention, a commercially available animal cell culture medium such as BME medium, MEM medium, DMEM medium, F10 medium, F12 medium, etc. It may replace with a component and may add and prepare the additive of this invention.
[0032]
In the present invention, Brix 5% or less in the culture medium, preferably Brix 0.5-3%, particularly preferably Brix 1-2% in the medium, the fish meat mixture extract or the enzymatic degradation product of the fish meat mixture is added to the culture medium. Added. In addition, the said density | concentration is a density | concentration which made the soluble solid content measured with the refractometer the index.
[0033]
In addition, the content of other components in the medium is 0.05-1500 mg / L for amino acids, 0.001-10 mg / L for vitamins, 0-200 mg / L for lipid factors, 1-20 g / L for energy sources, osmotic pressure regulation 0.1-10000mg / L for iron, 0.1-500mg / L for iron source, 1-10000mg / L for pH buffer, 0.00001-200mg / L for trace metal elements, 0-5000mg / L for surfactant, growth factor Is in the range of 0.05 to 10000 μg / L and nucleoside is in the range of 0.001 to 50 mg / L, and can be appropriately determined depending on the type of animal cells to be cultured, the type of desired protein, and the like.
[0034]
The pH of the medium varies depending on the cells to be cultured, but is generally pH 6.8 to 7.6, and in many cases pH 7.2 to 7.4.
The medium of the present invention is not particularly limited and can be used for suitably culturing various animal cells. For example, fusion represented by COS cells and CHO cells into which a desired antibody or physiologically active substance gene is incorporated by genetic engineering operations, or hybridomas such as mouse-human, mouse-mouse, mouse-rat, etc. that produce antibodies Cells can be cultured. In particular, it is suitable for CHO cell culture. Of course, the animal cell culture medium of the present invention can also be used when culturing animal cells to obtain a natural protein produced by the animal cells. In addition to the cells described above, BHK cells, HeLa cells It can also be used for culturing.
[0035]
Since culture conditions differ depending on the type of cells used, suitable conditions may be determined as appropriate. For example, in the case of CHO cells, it is usually cultured for 1-14 days at 30-39 ° C., preferably about 37 ° C., in an atmosphere with a CO ₂ concentration in the gas phase of 0-40%, preferably 2-10%. That's fine.
[0036]
In addition, as various culture apparatuses for animal cell culture, for example, fermenter tank culture apparatus, air lift culture apparatus, culture flask culture apparatus, spinner flask culture apparatus, microcarrier culture apparatus, fluidized bed culture Culture can be performed using an apparatus, a holofiber type culture apparatus, a roller bottle type culture apparatus, a filled tank type culture apparatus, or the like.
[0037]
By culturing in the animal cell culture medium of the present invention, the protein produced by the animal cells can be obtained in the medium. The production of protein in animal cells includes those that can be simply cultured, and those that require special operations, but these operations or conditions may be appropriately determined depending on the animal cells to be cultured. For example, in a CHO cell transformed with a vector containing a gene encoding a mouse-human chimeric antibody by genetic engineering, the culture is carried out under the conditions as described above for 1-14 days, preferably 7-10. The desired protein can be obtained in the medium in about days. By isolating and purifying this according to conventional methods (eg, Introduction to Antibody Engineering, Jinshokan, p.102-104; Affinity Chromatography Principles & Methods, Amersham Pharmacia Biotech, p.56-60, etc.) The desired protein can be obtained.
[0038]
Recombinant antibodies such as anti-human IL-6 receptor antibody (including chimeric antibodies, humanized antibodies, humanized antibodies), granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimuli Genetically modified proteins such as factor (GM-CSF), erythropoietin, interferon, interleukins such as IL-1 and IL-6, t-PA, urokinase, serum albumin, blood coagulation factor VIII and the like can be produced.
[0039]
【Example】
Examples for explaining the present invention in more detail are shown below, but the present invention is not limited to these examples. Various changes and modifications can be made by those skilled in the art, and these are also included in the scope of the present invention.
Example 1:
(1) Preparation of Enzyme Decomposition Product of True Meat Commercially available salmon was used as fish meat. Remove the internal organs, bones, heads, etc. from the fish meat, add 1500 g of water to 1000 g of minced cut, and incubate with 4 g of plant-derived papain for 1 hour at pH 6.0, 50 ° C. went. Next, after further enzymatic degradation with 4 g of mold-derived exopeptidase under the above conditions for 15 hours, the enzyme was inactivated by heating to 95 ° C. Thereafter, insoluble matters and oil were removed by centrifugation and filtration, and concentrated to prepare 500 g of an enzymatic decomposition product of the true meat (rice cake) of the present invention.
(2) Preparation of Visceral Enzymatic Degradation Products Commercial salmon was used as fish meat. Only the internal organs were taken out from the fish meat, and the extracted internal organs were treated in the same manner as the preparation of the enzymatic decomposition product of the true meat of (1) to prepare 500 g of the enzymatic decomposition product of the internal organs (sardine).
(3) Preparation of a mixture of visceral and meat digests
In the same manner as in (1) and (2), an enzyme degradation product was prepared in which viscera: meat were mixed at a ratio of 7: 3, 6: 4, 5: 5, 4: 6, 3: 7.
Example 2 Preparation of Medium As a basic animal cell culture medium (basic medium), a medium obtained by removing thymidine and hypoxatin from commercially available DMEM / F12 / medium (GIBCO BRL Product and Reference Guide, p357-358) What added these components was used.
[0040]
A medium obtained by removing thymidine and hypoxatin from a commercially available DMEM / F12 medium, with the following components added:
Ascorbic acid 30mg / L,
Deoxyadenosine (1H ₂ O) 10 mg / L,
Deoxycytidine 10 mg / L,
Deoxyguanosine 10mg / L,
Adenosine 5mg / L,
Cytidine 5mg / L,
Guanosine 5mg / L,
Uridine 5mg / L,
Ethanolamine (HCl) 4mg / L,
Pluronic F-68 1000mg / L,
Ferric chloride (6H ₂ O) 18.9mg / L
10 g / L or 17.5 g / L of the enzyme degradation product prepared in Example 1 was added to the medium (basic medium) in terms of solid content, respectively, and sterilized by filtration.
Example 3: Effect of visceral to true meat ratio on cell viability Using the human elongation factor-Iα promoter described in Example 10 of International Patent Application Publication No. WO92 / 197559, The test was carried out using a CHO cell line producing humanized PM-1 antibody (anti-human IL-6 receptor antibody) prepared according to the method described in Reference Example 2 of Kaihei 8-99902.
[0041]
The above-mentioned CHO cells (1.5 ×) were prepared in a medium in which the visceral: meat ratio prepared in Example 2 was changed (a medium obtained by adding 17.5 g / L of the enzyme degradation product prepared in Example 1 (4) to the basic medium). 10 ⁵ cell / ml) was added, and the cells were cultured for 12 days under an incubation condition of 37 ° C. and 5% CO ₂ using a shaker flask type cell culture apparatus.
[0042]
Subsequently, the cell viability after 7 days, 10 days, and 12 days from the start of the culture was measured. The results are shown in FIGS.
After 7 days of culture, the viability was high when the visceral: real ratios were 7: 3, 6: 4, 5: 5, 4: 6, and the viability was low at 3: 7.
[0043]
After 10 days of culture, the viability was high when the visceral: real ratio was 7: 3, 6: 4, and it was revealed that the cells were not alive at 4: 6, 3: 7.
After 12 days of culture, it was clarified that cells survived when the visceral: meat ratio was 6: 4, and cells did not survive at other ratios.
Example 4: Effect on antibody production amount The medium prepared in Example 2 of the present specification (basic medium + meat digestion product 4 g / L + visceral enzyme degradation product 6 g / L (ratio of viscera: meat 6: 4) ), CHO cells (1.5x10 ⁵ cell / ml) are added to basic medium + meat digestion product 10g / L, basic medium + visceral enzyme digestion product 10g / L), and shaker flask type cell culture device is used. The cells were cultured for 10 days under incubator conditions at 37 ° C. and 5% CO 2.
[0044]
Subsequently, the production amount of the antibody protein obtained from the medium was measured. The production amount was measured using reverse phase high performance liquid chromatography. The results are shown in FIG.
When cultured in a medium supplemented with a mixture of visceral and meat digests, it has a higher effect on the amount of antibody protein produced than when cultured in a medium supplemented with visceral or digested enzymes alone. Indicated. Therefore, it became clear that the viscera and the meat mixture were more effective than the meat alone or the viscera alone.
[0045]
From the above results, it became clear that the amount of antibody production was higher in the mixture of fish meat and true meat than in the internal organs of fish meat or just meat. In terms of cell viability, the effect is high when the ratio of viscera to meat is 7: 3 to 4: 6, higher at 7: 3 to 6: 4, and highest at 6: 4. There was found.
[0046]
【The invention's effect】
The medium for culturing animal cells of the present invention uses an enzyme degradation product of a mixture of fish internal organs and meat, or an extract of the mixture, so that expensive and highly variable protein such as fetal bovine serum is used. It is possible to cultivate animal cells stably without doing so. In addition, the medium of the present invention is superior in both cell viability and protein production as compared with a medium containing only fish internal organs or only meat. Furthermore, by culturing animal cells in the cell culture medium of the present invention, the risk of contamination with abnormal prions or viruses, which has become a problem in recent years, is eliminated, and safe biopharmaceuticals can be produced and provided.
[Brief description of the drawings]
FIG. 1 is a graph showing cell viability after 7 days when CHO cells are cultured in media containing various rates of visceral enzyme digest and fish enzyme digest in various ratios.
FIG. 2 is a graph showing cell viability after 10 days when CHO cells are cultured in a medium containing various rates of visceral enzyme digest of fish meat and authentic enzyme digest of meat.
FIG. 3 is a graph showing the cell viability after 12 days when CHO cells were cultured in media containing various rates of visceral enzyme digests and fish enzyme digests in various ratios.
FIG. 4 is a graph showing the effect on the amount of antibody production when CHO cells are cultured in a medium containing a visceral enzyme digest of fish meat, a digested meat digest or a mixture thereof.

Claims (18)

An additive to a serum-free medium for animal cell culture , comprising an enzyme degradation product of a mixture of fish viscera and meat, wherein the weight ratio of the viscera and meat is 75:25 to 40:60 .   The additive according to claim 1, wherein the enzyme degradation product of the mixture is obtained using proteinase and / or peptidase.   The additive according to claim 2, wherein the proteinase or peptidase is not derived from a mammal. The additive according to claim 2 or 3, wherein the enzyme degradation product of the mixture is obtained by subjecting the mixture or mixture extract to proteinase treatment and then peptidase treatment. The additive according to any one of claims 1 to 4, wherein a weight ratio between the viscera and the meat is 65:35 to 55:45. The additive according to claim 5 , wherein the weight ratio between the viscera and the meat is 60:40. The additive according to any one of claims 1 to 6 , wherein the fish meat is one or more selected from the group consisting of salmon, soda bonito, salmon, salmon, salmon and salmon. A method for producing an additive to an animal cell culture medium comprising the following steps:
(A) a step of mixing the internal organs and the true meat of fish meat in a weight ratio of the internal organs and the authentic meat of 75:25 to 40:60 , and (b) an enzymatic decomposition of the above mixture to obtain an enzymatic decomposition product as that of Engineering. A method for producing an additive to an animal cell culture medium comprising the following steps:
(A) the internal organs of the fish meat as obtained Ru engineering the by enzymatic degradation enzyme degradation product,
(B) a positive meat fish as enzymatic degradation and give Ru Industrial enzymatic degradation product, and (c) above (a) and material prepared by the process step of mixing the substance prepared in the above (b) step,
However, the said method whose weight ratio of the internal organs and the true meat in a process (a) and a process (b) is 75: 25-40: 60 . The method according to claim 9 , wherein the weight ratio between the viscera and the meat is 65:35 to 55:45. The method according to claim 10 , wherein the weight ratio of the viscera to the meat is 60:40. A serum-free medium for animal cell culture comprising the additive according to any one of claims 1 to 7 . Culture locations claim 12, wherein the medium for producing a desired protein by culturing the animal cell. For animal cell culture medium, amino acids, vitamins, lipid factors, energy sources, osmotic regulators, iron sources and claim 12 or 13 culture locations described comprises a pH buffering agent. Animal cells, culture locations according to any one of claims 12 to 14 gene in which the introduced which encodes a desired protein. Desired protein, culture ground of claim 15, wherein an antibody or physiologically active substance. Animal cells, culture locations according to any one of claims 12-16 which is a CHO cell. In the method for producing a desired protein by culturing animal cells, the culture is performed by enzymatic degradation of a mixture of fish viscera and meat having a weight ratio of viscera to meat of 75:25 to 40:60. A method for producing a protein, which comprises using a serum-free medium for animal cell culture, characterized by comprising a product .

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