JPH089968A - Medium characterized by containing n-butyric acid for culturing animal cell and method for culturing the animal cell - Google Patents

Abstract

PURPOSE:To obtain the subject medium easy in the separation and purification of proteins produced therein, capable of efficiently culturing recombinant animal cells, etc., capable of manifesting an antibody gene such as of mouse-human chimera antibody. CONSTITUTION:This medium is free from protein component, and contains n-butyric acid in a concentration of pref. 0.01-5mM and at least an iron component selected from iron citrate and iron sulfates and at least one kind selected from 2-ethanolamine, sodium selenite, taurine, linoleic acid and oleic acid. It is preferable that animal cells such as recombinant animal cells capable of manifesting antibody gene or animal fused cells capable of producing antibody be cultured in this medium.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a medium for culturing animal cells, a method for culturing animal cells using the medium, and a method for producing a gene recombinant product and the like.

[0002]

BACKGROUND OF THE INVENTION Since the production method of monoclonal antibody by Keller and Milstein in 1975,
The use of monoclonal antibodies has been active in a wide range of fields from medical fields such as diagnosis and treatment to industrial fields such as separation and purification of antigens. Monoclonal antibodies have already been widely put to practical use in the field of in-vitro diagnostics, and also partially in the therapeutic fields such as in-vivo diagnostics such as immuno-imaging and immunosuppressants. Further, active research and development are also being carried out for industrial uses such as reagents for purifying antigens such as proteins.

As a method for producing a monoclonal antibody,
Conventionally, the main method was to culture fused cells of mouse cells (for example, hybridoma) in mouse ascites fluid and obtain monoclonal antibody from the ascites fluid. Is becoming active.

Furthermore, regarding the application of antibodies to in-vivo diagnostic agents and therapeutic agents, the production of chimeric antibodies in which a part of a mouse monoclonal antibody is replaced with a human antibody has been actively conducted by applying genetic engineering techniques. These are mainly produced by recombinant animal cells into which the antibody gene has been introduced.

Since such a chimeric antibody is composed of parts derived from two or more animal species, it is more suitable to be produced by the cell culture method than in the body of a specific animal. In general, the antibody production by Escherichia coli is significantly lower than the conventional production of monoclonal antibodies by mouse hybridomas. On the other hand, in order to apply a monoclonal antibody to in-vivo diagnosis and treatment, it is essential to supply a large amount of a highly purified antibody in a stable manner, and an efficient culture production method therefor is desired. There is.

[0006]

The culturing of animal cells such as fused cells and recombinant animal cells by the cell culturing method involves culturing animal serum (often in many cases) on a basal medium consisting of amino acids, vitamins, sugars, salts and the like. Is fetal bovine serum),
Insulin, transferrin, 2-ethanolamine, sodium selenite, albumin, cell growth factors, etc. are used without serum (serum-free medium), but serum, insulin, cells Since growth factors and the like are very expensive, the cost of the medium itself is high, and as a result, the antibody produced is expensive.

Furthermore, known or unknown protein components contained in serum, insulin, cell growth factors, etc. must be finally separated from the produced protein having antibody activity, monoclonal antibody, etc. There is also. That is,
This is because when the produced chimeric antibody or the like is used as an in-vivo diagnostic agent or in-vivo therapeutic agent, it is necessary to completely remove contaminants, particularly protein components. Therefore, in the case of producing a chimeric antibody or the like by culturing recombinant cells or fused cells capable of expressing the antibody gene in a medium containing serum or the like, a combination of various purification methods that are usually used is complicated. Refining operation,
An operation for checking the degree of purification is required.

In recent years, in order to solve the above problems, a serum-free medium containing no protein component has been studied (Japanese Patent Application Laid-Open No. 4-91786, Japanese Patent Application No. 6-25125, etc.). However, the problem of low antibody productivity per cell occurs in these serum-free media. Especially when trying to produce an antibody or the like by culturing recombinant cells capable of expressing antibody genes or human-human hybridomas, etc., these are usually very low in productivity of the antibody, etc. It should be solved.

[0009]

[Means for Solving the Problems] The present inventors have improved the productivity by culturing a genetically modified cell producing a chimeric antibody and producing the chimeric antibody by simply adding it to a conventional serum-free medium. As a result of searching for substances that can be used, the present invention has been completed.

That is, the present invention is an animal cell culture medium containing n-butyric acid. The present invention also provides
A method for culturing animal cells, which comprises culturing animal cells in a medium containing n-butyric acid. Further, the present invention is
A method for producing a protein produced by an animal cell, which comprises culturing the animal cell in a medium containing n-butyric acid. Hereinafter, the present invention will be described in detail.

The medium of the present invention is characterized by containing a low molecular weight substance consisting of n-butyric acid, and the component other than the low molecular weight may be a commercially available basal medium used for ordinary animal cell culture. In the case of long-term culture or high-density culture, a medium containing amino acids and vitamins at a high concentration, for example, a commercially available E-RDF medium (manufactured by Kyokuto Pharmaceutical Co., Ltd.), RP
MI1640 (manufactured by GIBCO), DMEM (GIBCO)
And F-12 (manufactured by GIBCO) in a ratio of 2: 1: 1 and a medium in which DMEM and F-12 are mixed in a ratio of 1: 1. In these serum-free media, at least iron components such as iron citrate and iron sulfate, 2-ethano-
It is preferable to add ruamine, sodium selenite, taurine, linoleic acid, oleic acid, or the like.

The present invention is a conventional medium (medium containing serum)
It can be applied to any of the serum-free media described above,
Considering the purification of the produced antibody and the like, a serum-free medium containing no protein component is particularly preferable.

The concentration of n-butyric acid in the medium depends on the cell, but is preferably 0.1 to 5 mM, particularly 0.25 to
0.5 mM is desirable.

By using the animal cell culture medium of the present invention containing n-butyric acid as an additive component, the desired protein can be efficiently produced. For example, it is particularly preferably used for recombinant animal cells capable of expressing antibody genes and animal fused cells capable of producing antibodies. Examples of the animal cell include, for example, animal cells including human, which produce a special protein, mouse-
Vector containing gene encoding human chimeric antibody
Myeloma cells, COS cells or CHO transformed with
Examples thereof include cells, and fused cells represented by hybridomas such as mouse-human, mouse-mouse, mouse-rat, etc. capable of producing monoclonal antibodies.

By culturing the above cells in the medium of the present invention, a desired protein such as an antibody can be produced.

[0016]

INDUSTRIAL APPLICABILITY By using the medium for culturing animal cells of the present invention, recombinants capable of expressing antibody genes such as mouse-human chimeric antibodies, which were difficult to mass-produce because the expression amount of the original product was very low. It is possible to efficiently culture animal cells and the like. Further, n-butyric acid can be applied not only to a serum medium but also to a serum-free medium or a protein-free medium, is inexpensive and very easily available, and has a small molecular weight and is desirable as a component constituting a serum-free medium or a protein-free medium. .

As described above, by using the medium of the present invention, the productivity of the target substance per cell can be significantly increased,
When mass-cultivating a recombinant animal cell capable of expressing an antibody gene such as a mouse-human chimeric antibody to produce a substance, it is possible to significantly reduce the cost of raw materials such as a medium,
And high yield can be expected.

Furthermore, when the present invention is applied to a serum-free medium or a protein-free medium, separation of proteins such as produced antibodies,
Purification can be performed easily. Therefore, it is possible to reduce the production cost of the produced protein such as antibody,
It is also possible to prevent a decrease in the yield of the target protein that may occur during purification.

[0019]

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 Mouse myeloma HCNd8A expressing a mouse-human chimeric Nd2 antibody subcultured in E-RDF medium (manufactured by Kyokuto Pharmaceutical) containing 10% (w / v) fetal bovine serum (Japanese Patent Application No. 6- Two
131), 10 μM taurine, 50 μM ferric citrate, 25 nM sodium selenite, 100 μM 2-ethanolamine, 15 μM sodium pantothenate, 0.5 μg /
ml linoleic acid, 0.5 μg / ml tocopherol acetate,
N-butyric acid was added to an E-RDF medium (hereinafter referred to as medium A) containing 0.5 μg / ml egg yolk lecithin and 0.05 μg / ml retinoyl acetate so that the final concentration shown in FIG. 1 was obtained,
Seeding the above culture at a cell concentration of 5 × 10 ⁵ cells / ml,
Culturing was started under the conditions of an incubator at 37 ° C. and 5% CO ₂ , and the cells were cultured for 3 days, and the number of cells, cell viability, glucose
The consumption rate and chimeric antibody concentration were measured. The result is shown in Figure 1.
Shown in.

As a result, when 0.5 mM of n-butyric acid was added,
Although the consumption rate of the substrate (glucose) was suppressed, the production amount of antibody was improved about 3-fold.

Example 2 In order to analyze in detail the effects of n-butyric acid on cell growth, substrate (glucose) consumption rate, and antibody productivity, the following experiment was conducted in the presence or absence of n-butyric acid. It was The medium A was used, and the concentration of n-butyric acid added was 0.5 mM. 2 x 10
⁵ cells / ml of cells were seeded and cultured for 6 days, and the number of cells, cell viability, glucose concentration, glucose consumption rate, chimeric antibody productivity and total antibody production were measured. The results are shown in FIGS.

As shown in FIGS. 2 and 3, the growth of cells was suppressed by adding n-butyric acid, but no significant difference was observed in the survival rate.

The results of culturing for 6 days are as shown in FIGS.
The amount of antibody produced was increased by about 2.3 times by the addition of n-butyric acid as compared with the case of no addition.

As shown in FIGS. 6 and 7, by adding n-butyric acid,
It was suggested that cell division was suppressed by n-butyric acid, such as a decrease in glucose consumption rate. Also, n-
The addition of butyric acid more than doubled the antibody productivity.

The specific antibody productivity (CSP) per cell is shown in FIG. The addition of n-butyric acid improved CSP by about 2 to 4 times, indicating that n-butyric acid is a very effective drug for improving antibody productivity per cell. .

From the results of this Example, it was confirmed that addition of 0.5 mM n-butyric acid suppresses cell growth and substrate consumption rates, but conversely improves specific antibody productivity.
-It became clear that butyric acid improves the production efficiency.

Example 3 In order to mass-produce antibodies, continuous production of chimeric antibodies using a culture device was examined using a hollow fiber type culture device.

Culture device (culture flow T / C-5
0, manufactured by Asahi Medical Co., Ltd.)
By using chimeric Nd2 antibody-expressing recombinant cells HCNd8
A was cultured in the presence or absence of n-butyric acid, and the production amount of the produced chimeric antibody was compared.

Each of 2 × 10 ⁷ cells was inoculated into a hollow fiber bioreactor and cultured in a medium A containing 3% fetal calf serum for about 1 week, and then the fetal calf serum concentration was adjusted to 1
%, 0.1%. Culturing was carried out under the conditions of 37 ° C. and 5% CO ₂ , the medium circulation flow rate was gradually increased, and from the 11th day, the medium circulation flow rate was 10 ml / min. The culture was continued for about 2 months, and the production amounts of chimeric antibodies were compared. The concentration of the added n-butyric acid was set to 0.25 mM in consideration of suppression of cell growth. The results are shown in FIGS.

As shown in FIGS. 9 and 10, the total antibody production was about 7.4 mg in the absence of n-butyric acid in the culture for 2 months, but in the presence of 0.25 mM n-butyric acid, the total antibody production was about 18 mg. The antibody production amount was achieved, and the antibody productivity was improved by about 2.4 times.
Also, the antibody productivity per day was 0.31 mg / day at maximum in the absence of n-butyric acid, but was 0.73 mg / day at maximum in the presence of n-butyric acid. Admitted.

[Brief description of drawings]

FIG. 1 shows the results of culturing HCNd8A in Example 1. GCR indicates glucose consumption for 3 days (black circle), and MoAb indicates antibody concentration in the culture supernatant after 3 days (white circle). The n-butyric acid concentration on the horizontal axis is shown on the logarithmic axis.

FIG. 2 shows HCNd8 in Example 2 in the absence of butyric acid.
The result when A was cultured is shown. Cell indicates the number of viable cells (black circles), and Viability indicates cell viability (white circles). The horizontal axis represents the number of culture days after the culture was started using the medium A.

FIG. 3 shows HCNd8A in the presence of butyric acid in Example 2.
The result when culturing is shown. Cell indicates the number of viable cells (black circles), and Viability indicates cell viability (white circles). The horizontal axis represents the number of culture days after the culture was started using the medium A.

FIG. 4 HCNd8 in the absence of butyric acid in Example 2.
The result when A was cultured is shown. Glucose indicates glucose concentration (black triangle), Total product
t indicates the total chimeric antibody production (open triangle). The horizontal axis represents the number of culture days after the culture was started using the medium A.

FIG. 5: HCNd8A in the presence of butyric acid in Example 2.
The result when culturing is shown. Glucose is gluco-
Density (black triangle), Total product
Indicates the total chimeric antibody production (open triangle). Horizontal axis is medium A
The number of culture days after the start of culture is shown.

FIG. 6 shows HCNd8 in the absence of butyric acid in Example 2.
The result when A was cultured is shown. GCR shows glucose consumption for 3 days (black square), productivit
y indicates the total chimeric antibody production (open square). The horizontal axis represents the number of culture days after the culture was started using the medium A.

FIG. 7 shows HCNd8A in the presence of butyric acid in Example 2.
The result when culturing is shown. GCR is glucose for 3 days
Consumption (black square), productivity
Indicates the total chimeric antibody production (open squares). Horizontal axis is medium A
The number of culture days after the start of culture is shown.

FIG. 8 shows HCNd8 in the absence of butyric acid in Example 2.
The result when A was cultured is shown. CSP is 1 for each living cell
The amount of antibody produced per hour is shown. Black circles show results in the absence of butyric acid, and black squares show results in the presence of butyric acid. The horizontal axis represents the number of culture days after the culture was started using the medium A.

FIG. 9: Culture made by Asahi Medical in Example 3
The result when HCNd8A was cultured in Flo-T / C-50 in the absence of butyric acid for about 2 months is shown. productiv
“Itity” indicates the antibody productivity per day (black triangle), To
Tal product indicates the total chimeric antibody production (black circle). The horizontal axis represents the number of culture days after the culture was started using the medium A.

FIG. 10 shows the results of culturing HCNd8A in Example 3 in the presence of butyric acid in Culture Flo-T / C-50 manufactured by Asahi Medical for about 2 months. productiv
“Itity” indicates the antibody productivity per day (black triangle), To
Tal product indicates the total chimeric antibody production (black circle). The horizontal axis represents the number of culture days after the culture was started using the medium A.

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Claims (16)

[Claims] 1. A medium for animal cell culture, which contains n-butyric acid. 2. The animal cell is a recombinant animal cell capable of expressing an antibody gene or an animal fusion cell capable of producing an antibody,
The medium for animal cells according to claim 1. 3. The concentration of n-butyric acid is 0.1-5 mM.
The medium for animal cells according to claim 1. 4. In addition to n-butyric acid, at least an iron component selected from iron citrate or iron sulfate, and one selected from 2-ethanolamine, sodium selenite, taurine, linoleic acid and oleic acid. The animal cell culture medium according to claim 1, comprising the above components. 5. The animal cell culture medium according to claim 1, which does not contain a protein component. 6. A method for culturing animal cells, which comprises culturing animal cells in a medium containing n-butyric acid. 7. The culture method according to claim 6, 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. 8. The culture method according to claim 6, wherein a medium containing 0.1 to 5 mM n-butyric acid is used. 9. In addition to n-butyric acid, at least an iron component selected from iron citrate or iron sulfate, and one selected from 2-ethanolamine, sodium selenite, taurine, linoleic acid and oleic acid. The culture method according to claim 6, wherein a medium containing the above components is used. 10. The culture method according to claim 6, wherein a medium containing no protein component is used. 11. A method for producing a protein produced by an animal cell, which comprises culturing the animal cell in a medium containing n-butyric acid. 12. The method according to claim 11, 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. 13. The method according to claim 11, wherein the animal cell is a recombinant animal cell capable of expressing an antibody gene and is a protein chimera antibody produced. 14. The production method according to claim 11, wherein a medium containing 0.1 to 5 mM n-butyric acid is used. 15. An iron component selected from at least iron citrate or iron sulfate, in addition to n-butyric acid, and one selected from 2-ethanolamine, sodium selenite, taurine, linoleic acid and oleic acid. The production method according to claim 11, wherein a medium containing the above components is used. 16. The production method according to claim 11, wherein a medium containing no protein component is used.