JPH07231783A - Protein-free medium for culture of animal cell and culture method - Google Patents

Abstract

PURPOSE:To obtain the subject medium containing tocopherol, retinol, lecithin and/or sodium pantothenate, useful for culture of not only a hybridoma but wider-ranging animal cells and suitable for along-term mass-culture in a cell culture equipment. CONSTITUTION:This medium contains one or more kinds of low-molecular substances selected from a group of tocopherol (tocopherol pcetate is included), retinol (retinol acetate is included), lecithin (yolk lecithin or soybean lecithin) and sodium pantothenate. An animal cell, e.g. a recombinant animal cell capable of manifesting an antibody gene or an animal fused cell capable of producing an antibody can be reproducibly cultured without requiring fetal bovine serum, etc.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a protein-free medium for culturing animal cells and a culture method for producing a monoclonal antibody.

[0002]

BACKGROUND OF THE INVENTION Since the technology for producing monoclonal antibodies was developed by Keller and Milstein (Nature, 256, 495, 1975) in 1975, many monoclonal antibodies against various antigens have been produced. It was Monoclonal antibodies are in vitro diagnostics, internal diagnostics, therapeutics,
It is being used as a reagent for affinity purification, and some have already been put to practical use, and research and development towards the practical use of some have been actively conducted.

[0003] Conventionally, production of a monoclonal antibody in which a fused cell of mouse cells (for example, a hybridoma) or a fused cell of a rat cell and a mouse cell is cultured in ascites fluid of a mouse to obtain a monoclonal antibody from the ascites fluid. Although the method was the main focus, in recent years it is shifting to cell culture. Further, human cells other than the above fused cells, fused cells of mouse cells and human cells, such as fused cells of mouse cells and human cells, which are difficult to culture in ascites of mice, and recombinants in which an antibody gene has been introduced using genetic engineering technology Animal cells are cultured by the cell culture method.

[0004]

The culturing of animal cells such as fused cells and recombinant animal cells by the cell culturing method has hitherto been carried out by using animal serum in a basal medium composed of amino acids, vitamins, sugars and salts. (In most cases, fetal bovine serum) or a medium supplemented with components purified from serum is used. However, serum, especially fetal bovine serum, is expensive and has a large variation in quality. There is a problem in reproducibility of culture results. In the case of producing a monoclonal antibody or a protein having antibody activity by culturing a fused animal typified by a hybridoma or a recombinant animal cell capable of expressing an antibody gene, the above-mentioned cost problem is, of course, produced. The variation in sex becomes a problem.

Known or unknown protein components contained in serum pose a problem when purifying a monoclonal antibody or the like produced after completion of culture. This is because when the produced monoclonal antibody or the like is used as an in-vivo diagnostic agent or in-vivo therapeutic agent, it is necessary to completely remove contaminants, especially protein components. Therefore, in the case of producing a monoclonal antibody or the like by culturing a recombinant animal cell or the like capable of expressing a fused cell or an antibody gene in a medium containing serum, various purification methods usually used are combined, A complicated purification operation is required.

As a serum-free medium, a medium to which insulin, transferrin, 2-ethanolamine, sodium selenite, albumin, cell growth factor, etc. are added may be used, but it is changed to contain a protein component. However, the same problem as described above occurs when the manufactured monoclonal antibody or the like is purified. Moreover, since insulin and cell growth factors are very expensive, there is a cost problem.

Recently, a medium containing no protein component has been reported (Japanese Patent Laid-Open No. 4-91786, Toyoda et al., Agric. Biol. Che.
m., 55, 1631, 1991 or Shinmoto et al., Biotechn.
ol. Lett., 13: 683, 1991, etc.), an attempt has been made to solve the problem of a medium containing a protein component. However, even in these protein-free media, it is difficult to culture depending on the type of animal cells, and the culture efficiency (growth of animal cells) is poor as compared with media containing serum or protein components,
Further, when performing long-term mass culture using a cell culture device, there were problems such as the cell viability gradually decreasing and the cell number decreasing.

There is a problem to be further improved.

[0009]

Means for Solving the Problems The present inventor has conducted extensive studies on a protein-free medium previously reported for culturing a hybridoma producing a monoclonal antibody and producing the monoclonal antibody. By adding several low molecular weight components to the above, it is possible to apply to the culture of more animal cells without the need to add protein components, and to find a protein-free medium suitable for long-term mass culture using a cell culture device. The present invention was started and the present invention was completed.

That is, the present invention is a protein-free medium for animal cell culture, which contains one or more low-molecular substances selected from the group consisting of tocopherol, retinol, lecithin and sodium pantothenate and does not contain a protein component. Further, the present invention is characterized by culturing animal cells in a protein-free medium containing at least one low-molecular substance selected from the group consisting of tocopherol, retinol, lecithin and sodium pantothenate, and containing no protein component. This is a method for culturing animal cells. The present invention further comprises tocopherol, retinol, lecithin and sodium pantothenate containing one or more low-molecular substances selected from the group consisting of culturing animal cells in a protein-free medium containing no protein component. It is a method for producing a protein produced by an animal cell. The present invention will be described in detail below.

The medium of the present invention comprises one or more low molecular weight substances selected from the group consisting of tocopherol (including tocopherol acetate), retinol (including retinol acetate), lecithin (egg yolk lecithin or soybean lecithin) and sodium pantothenate. Contains and does not contain protein components. The components other than the low molecular weight substances are proteins, cell growth factors, hormones, steroids, and other protein-free components, and components of conventional protein-free media such as basal media consisting of vitamins, amino acids, sugars and salts. It can be the same. For example, commercially available E-RDF medium (manufactured by Kyokuto Pharmaceutical), R
PMI-1640 (manufactured by GIBCO), DMEM (GIB
CO) and F-12 (GIBCO) at a ratio of 2: 1: 1, and DMEM and F-12 at 1: 1.
In the medium mixed in the ratio of, iron components such as iron citrate and iron sulfate to construct a protein-free medium, 2-ethanolamine, sodium selenite, taurine, linoleic acid or oleic acid can be added. However, it is not limited to these.

More specifically, the components contained in the protein-free medium of the present invention include, for example, tocopherol (including tocopherol acetate), retinol (including retinol acetate), lecithin (egg yolk lecithin or soybean lecithin) and sodium pantothenate. In addition to one or more selected from the group consisting of, preferably three or more, and particularly preferably four low molecular weight substances, ferric citrate, 2-ethanolamine, sodium selenite, taurine, L-
Alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamine, L-
Glycine glutamate, L-histidine, L-hydroxyproline, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L- Tyrosine, L-valine, glutathione, para-aminobenzoic acid, biotin, calcium pantothenate,
Choline tartrate (or choline chloride), folic acid, inosito-
, Nicotinic acid amide, pyridoxal hydrochloride, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, vitamin B1
2, lipoic acid, hypoxanthine, putrescine dihydrochloride,
Thymidine, linoleic acid (or oleic acid), sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, sodium monohydrogen phosphate, sodium pyruvate,
Copper sulfate, D-glucose, ferrous sulfate, zinc sulfate, HE
A medium containing PES and sodium hydrogen carbonate can be exemplified. The preferable examples of the medium of the present invention may contain antibiotics such as streptomycin and potassium penicillin G, and a pH indicator such as phenol red.

Each of the low molecular weight components contains 0.1 to 0.1 tocopherol (including tocopherol acetate) in the medium.
1.0, preferably about 0.2 to 0.5 μg / ml, retinal (including retinol acetate) 0.01 to
0.2, preferably about 0.05 to 0.2 μg / mL,
Lecithin (egg yolk lecithin or soy lecithin) is 0.1
2.0 preferably 0.3 to 1.0 μg / mL, sodium pantothenate 5 to 50, preferably 10 to 20
It may be added at a concentration of about μM. For example,
Tocopherol (and / or tocopherol acetate), retinol (and / or retinol acetate), lecithin (egg yolk lecithin and / or soybean lecithin) can be added to an ethanol solution, for example, tocopherol acetate 100 mg / mL, retinol acetate 10
mg / mL and egg yolk lecithin 100 mg / mL were dissolved to prepare a high-concentration additive solution, while sodium pantothenate was prepared as a high-concentration aqueous solution of about 300 mM, and these were added to the above concentrations. Is also good.

The low molecular weight substance can achieve the effect of compensating for the deficiency of the protein component by containing one or more selected from the group consisting of tocopherol, retinol, lecithin and sodium pantothenate, but the effect becomes weak. It is easy, and the animal cells to be cultured may be limited. Therefore, it is more preferable to add three or more kinds selected from the above group, but it is most preferable to add all the low molecular weight substances of the above group to achieve a higher effect for a larger number of kinds of animal cells. .

According to the protein-free medium of the present invention, animal cells can be preferably cultivated without any particular limitation. However, considering the effect of the present invention that facilitates the purification of the protein secreted in the medium by not containing the protein organism, the protein-free medium of the present invention is the animal cell prepared for the purpose of producing the protein. It is particularly preferably used for recombinant animal cells capable of expressing antibody genes and animal fused cells capable of producing antibodies. As an animal cell, for example, an animal cell such as a human that produces a special protein, or a vector containing a gene encoding a mouse-human chimeric antibody by genetic engineering is transformed.
Examples thereof include fused COS cells and CHO cells, and fused cells represented by hybridomas such as mouse-human, mouse-mouse, mouse-rat, etc. capable of producing a monoclonal antibody.

It is possible to culture the above-mentioned animal cells by placing the desired cells in the protein-free medium of the present invention and keeping them under appropriate conditions. The culture conditions are
It may be determined depending on which cell is to be cultured,
For example, in the case of a mouse-rat hybridoma, it is usually 3
It can be exemplified that the temperature is maintained at about 7 ° C. Further, it is also possible to culture hybridomas using various culturing devices for culturing animal cells, for example, fermenter-type tank culturing devices, perfusion-type culturing devices, microcarrier-type culturing devices, hollow fiber-type culturing devices, and the like. it can.

By carrying out the culture operation, the protein produced by the animal cell can be obtained in the medium.
For production of protein in animal cells, there are some which require only a simple culture and those which require a special operation (for example, induction of protein expression in a transformant). The conditions and the like may be appropriately determined depending on the animal cell to be cultured. For example, in a mouse-rat hybridoma, the desired protein can be obtained in the medium in about 5 days by culturing under the conditions described above (although it varies depending on various conditions).

[0018]

The protein-free medium for animal cells of the present invention contains at least one low-molecular substance selected from the group consisting of tocopherol, retinol, lecithin and sodium pantothenate, so that it is expensive such as fetal bovine serum. It is possible to culture animal cells without using a protein component having a large variation in quality. Therefore, it is cheaper than conventional media containing protein components such as fetal bovine serum,
Moreover, the medium has good reproducibility of culture results. As a result, according to the culture method of the present invention, it is possible to perform culture with good reproducibility and to always obtain a constant culture result. In addition, the method for producing a protein of the present invention makes it possible to obtain a desired protein at a low yield and in a constant yield.

In particular, in the method for producing a protein of the present invention, after carrying out the culture operation, the purification operation for the produced protein can be simplified. For example, when the produced monoclonal antibody or the like is used as an in-vivo diagnostic agent or in-vivo therapeutic agent, it is necessary to completely remove contaminants, especially protein components, but the protein-free medium of the present invention that does not originally contain protein components If used, contaminants can be removed by a relatively easy purification operation.

According to the present invention, culturing is carried out in a conventional medium that does not contain protein components such as fetal bovine serum and one or more low molecular weight substances selected from the group consisting of tocopherol, retinol, lecithin and sodium pantothenate. It is possible to achieve good culturing of animal cells that have been difficult to grow and animal cells such as hybridomas that have been poorly grown in such conventional media. Therefore, when a hybridoma or the like is cultured, it becomes possible to produce a monoclonal antibody or the like with a higher yield and maintaining the yield for a long period of time.

Further, as described in Examples, in the production of monoclonal antibodies by continuous culture using a cell culture device, one or more low molecular weight substances selected from the group consisting of tocopherol, retinol, lecithin and sodium pantothenate are used. It is possible to achieve a good culture result that can maintain a high production amount for a long period of time as compared with a conventional medium that does not contain it.

[0022]

EXAMPLES Examples will be shown below for illustrating the present invention further in detail, but the present invention is not limited to these examples.

Example 1 First, the individual components of tocopherol acetate, retinol acetate, egg yolk lecithin, and sodium pantothenate were examined.

Hybridomas P7 and H6 of mouse cells subcultured in E-RDF medium (manufactured by Kyokuto Pharmaceutical Co.) containing 8% fetal bovine serum were mixed with 10 μM taurine, 50 μM ferric citrate, 25 nM sodium selenite, 100μ
The cells were cultured in an E-RDF medium containing M2-ethanolamine and 0.5 μg / mL linoleic acid (hereinafter referred to as medium A) for 7 days. Tocopherol acetate (manufactured by Sigma) was dissolved in ethanol at 1 mg / ml and added to medium A so that the final concentration shown in Table 1 was obtained, and the culture was made to have a cell concentration of 5 × 10 ⁴ cells / mL. Was inoculated, the culture was started under the incubator conditions of 37 ° C. and 5% CO ₂ , and the cells were cultured for 4 days to measure the viable cell concentration. The results are shown in Table 1. From Table 1 it can be seen that good growth is achieved with tocopherol acetate and its optimal concentration is 0.5 μg / mL.

[0025]

[Table 1]

Example 2 Example 1 was repeated except that a medium prepared by dissolving retinol acetate (manufactured by Sigma) in ethanol at 1 mg / ml was added to the medium A to give the final concentration shown in Table 2.
The same operation was performed. The results are shown in Table 2. From Table 2,
It can be seen that good growth is achieved with retinol acetate and its optimal concentration is 0.05 μg / mL.

[0027]

[Table 2]

Example 3 Similar to Example 1 except that a solution prepared by dissolving egg yolk lecithin (manufactured by Sigma) in ethanol at 1 mg / ml was added to the medium A so that the final concentration shown in Table 3 was obtained. The operation was performed. The results are shown in Table 3. From Table 3, it can be seen that good growth is achieved by egg yolk lecithin and its optimum concentration is 0.5 μg / mL.

[0029]

[Table 3]

Example 4 The same operation as in Example 1 was performed, except that a 100 mM aqueous solution of sodium pantothenate (Tokyo Kasei) was added to the medium A so that the final concentration shown in Table 4 was obtained. The results are shown in Table 4. From Table 4 it can be seen that better growth is achieved than sodium pantothenate and its optimal concentration is 15 μM.

[0031]

[Table 4]

Example 5 Hybridomas Q5 and C4 of mouse cells subcultured in E-RDF medium (manufactured by Kyokuto Pharmaceutical Co., Ltd.) containing 8% fetal bovine serum were added to medium A and medium A shown in Example 1. 1/20000 volume and 3 of ethanol solution containing tocopherol acetate 100 mg / mL, retinol acetate 10 mg / mL, and egg yolk lecithin 100 mg / mL.
1/2000 of 00 mM sodium pantothenate solution
0 volume was added, 0.5 μg / mL tocopherol acetate, 0.05 μg / mL retinol acetate,
Culture was performed using a medium (hereinafter referred to as medium B) in which 0.5 μg / mL egg yolk lecithin and 15 μM sodium pantothenate were used. They were seeded in dishes in medium A or B at a cell concentration ^{5 × 10 4 cells / mL,} 37 ℃, _of 5% CO ₂
Culturing was started under incubator conditions, and the cells were cultured for 7 days to measure the viable cell concentration.

Results for hybridoma Q5 are shown in FIG.
The results for C4 are shown in FIG. It can be seen from FIGS. 1 and 2 that hybridomas Q5 and C4 both showed better growth in medium B than medium A.

Example 6 Continuous production of a monoclonal antibody using a culture device was tried using a hollow fiber type cell culture device.

Culture device (Culture Flow T / C-11
00, manufactured by Asahi Medical Co., Ltd., and a hollow fiber bioreactor (Cellfirm BR1930, manufactured by Unisin Fibertech) was attached to the medium A used in Example 5.
Hybridomas P7 of mouse cells were cultured using B and B, and the production amounts of the produced monoclonal antibodies were compared.

More specifically, 6 × 10 ⁸ cells each were seeded in a hollow fiber bioreactor, cultured for 2 days in medium A containing 3% fetal bovine serum, and then switched to medium A or medium B for culturing. Continued. Culture is 3
At 7 ° C, gradually increase the medium circulation flow rate, medium supply rate, and oxygen supply rate, and from the 10th day, increase the medium circulation rate to 320 mL.
/ Min, medium supply 3.2 L / day, oxygen supply 40 m
L / min, and air supply rate was 40 mL / min.

As a result, in the medium A, about 1 day from 1
Although the production amount of about 60 mg / day was achieved, the production amount varied greatly, and the production amount gradually decreased from about 35 days after the start of the culture. On the other hand, in the medium B, the production amount is stable and 160 to 180 m over 10 to 62 days.
Production of as much as g / day was achieved, with a total of approximately 9.
It was possible to produce 6 g of monoclonal antibody. By the way, the total amount of production in 62 days using medium A is
It was about 8.5 g. Thus, it can be seen that medium B can maintain high and constant productivity in long-term culture.

The results obtained when the medium B was used are shown in FIG.

[Brief description of drawings]

FIG. 1 shows the results when hybridoma Q5 was cultured in Example 5. In the figure, the vertical axis represents the number of cells, and the horizontal axis represents the number of culture days since the culture was started using the medium A or B.

FIG. 2 shows the results of culturing hybridoma C4 in Example 5. In the figure, the vertical axis represents the number of cells, and the horizontal axis represents the number of culture days since the culture was started using the medium A or B.

FIG. 3 shows that the hybridoma P was added to medium B in Example 6.
The result when cultured using is shown. In the figure, the vertical axis represents the number of cells, and the horizontal axis represents the number of culture days since the culture was started using the medium B.

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Claims (13)

[Claims] 1. A protein-free medium for animal cell culture containing one or more low-molecular substances selected from the group consisting of tocopherol, retinol, lecithin and sodium pantothenate, and containing no protein component. 2. The protein-free medium for animal cell culture according to claim 1, wherein the animal cell is a recombinant animal cell capable of expressing an antibody gene or an animal fused cell capable of producing an antibody. 3. The culture medium according to claim 1, which contains at least three kinds of low molecular weight substances selected from the group consisting of tocopherol, retinol, lecithin and sodium pantothenate. 4. The medium according to claim 1, which contains tocopherol, retinol, lecithin and sodium pantothenate. 5. A method for culturing animal cells in a protein-free medium containing at least one low-molecular substance selected from the group consisting of tocopherol, retinol, lecithin and sodium pantothenate and containing no protein component. Method for culturing animal cells. 6. The culture method according to claim 5, wherein the animal cell is a recombinant animal cell capable of expressing an antibody gene or an animal fused cell capable of producing an antibody. 7. The medium is tocopherol, retinol,
The culture method according to claim 5, which comprises three or more low-molecular substances selected from the group consisting of lecithin and sodium pantothenate. 8. The medium is tocopherol, retinol,
The culture method according to claim 5, which comprises lecithin and sodium pantothenate. 9. A method for culturing animal cells in a protein-free medium containing at least one low-molecular substance selected from the group consisting of tocopherol, retinol, lecithin and sodium pantothenate and containing no protein component. A method for producing a protein produced by an animal cell. 10. The method according to claim 9, wherein the animal cell is a recombinant animal cell capable of expressing an antibody gene, and the protein produced is a protein having antibody activity. 11. The method according to claim 9, wherein the animal cell is a fused animal cell capable of producing an antibody, and the protein produced is a monoclonal antibody. 12. The method according to claim 9, wherein the medium contains three or more low-molecular substances selected from the group consisting of tocopherol, retinol, lecithin and sodium pantothenate. 13. The medium is tocopherol, retinol,
The method according to claim 9, which comprises lecithin and sodium pantothenate.