JPS63196268A - Transformant cell capable of being subjected to passage multiplication in serum-free medium, breeding thereof and production of protein by said cell - Google Patents

Abstract

PURPOSE:To produce useful protein such as gamma interferon, by cultivating a transformant cell having a transduced gene to code the useful protein, capable of being subjected to passage multiplication, in a serum-free medium. CONSTITUTION:A gene to code useful protein such as gamma interferon, tissue polasminogen activator, lymphotoxin, etc., is transduced to a host cell such as CHO cell, HeLa cell, etc., capable of being subjected to passage multiplication and the gene is selected in the serum-free medium to breed a transformant cell capable of being subjected to passage multiplication. Then the transformant cell is cultivated in the serum-free medium to produce the useful protein.

Description

Detailed Description of the Invention "Field of Industrial Application" The present invention relates to transformed cells into which a gene encoding a useful protein has been introduced and which can be subcultured in a serum-free medium to produce the useful protein, and the breeding of such cells. The present invention relates to a method and a method for producing useful proteins using the cells.

"Prior Art" Mass culture of animal cells has been carried out for the production of viruses and vaccines, as well as for the production of interferons and monoclonal antibodies from leukocytes and hybridomas. In addition, with the recent advances in genetic recombination technology, unlike proteins produced using bacteria such as Escherichia coli and Bacillus subtilis, proteins are produced as natural forms that exist in living organisms, such as glycosylated silanes and various modifications. Because of these many advantages, the production of useful proteins using animal cells as hosts is attracting attention, and the importance of large-scale culture technology is increasing.

One of the problems in producing useful substances by mass culturing animal cells is that the density of animal cells reached is low. One solution to this problem is a high-density culture method using medium perfusion, and various devices have been developed (W, R, Tol
bert et al., InVitro, Vol. 17, No. 10, p. 885, 1981, JP-A-6O-948.
2), attempts have also been made to improve cell density by searching for rate-limiting components in the culture medium and supplementing rate-limiting amino acids, etc. (Japanese Patent Laid-Open No. 60-54677).

The second problem is that the growth of animal cells requires 5 to 20% serum in a basal medium containing sugars, amino acids, vitamins, nucleic acids, etc. Serum is not only expensive and accounts for most of the cost of the culture medium, but also contains a wide variety of proteins, which complicates the purification of the target protein. Furthermore, the quality of serum differs depending on the loft, and it has a large effect on cell proliferation, and it is also possible that it may become a source of virus and mycoplasma contamination.

On the other hand, since the role of serum is to supply hormones and growth factors to cells, many attempts have been made to eliminate serum [G, 5atOs Analytical Biochemistry
) Vol. 102, p. 255, 1980], hormones and growth factors used in serum-free culture include hydrocortiscin, 'T3, insulin, transferrin, epidermal growth factor, fibroblast growth factor, platelet-derived For example, growth factors, etc., but the types and actions are described in Cell Growth Factors (Japanese Society for Tissue Culture, Line, Breakfast Shoten 1984).
(2013) is detailed in the following. In addition, new growth factors are being searched for, including a growth promoting factor extracted from adult bovine serum (Japanese Patent Application Laid-Open No. 58-206528), a growth promoting factor derived from horse serum (Japanese Patent Application Publication No. 61-63281), and Growth factors derived from ``Fermentation and Industry'' Vol. 43, No.
6, 1985) etc. have been reported.

The growth factors required for serum-free culture differ depending on the individual cells, and many serum-free media containing various growth factors have been reported.For example, CHO cells require insulin, transferrin, selenium [in vitro, cellular and・Developmental Biology (In
Vitro, Celural & Developom
21, No. 10, 58
8 Shin, 1985), He1a cells are treated with insulin, hydrocortiscin, transferrin, epidermal growth factor, and fibroblast growth factor [Proceedings of the National Academy of Sciences (Proc.
Natl, ^cad, Sci, USA) 75 volumes,
2, p. 901, 1978), BHK cells contain insulin, fibroblast growth factor, phipronectin, transferrin, and oleic acid [Journal of Cellular Physiology (Journal of Cellular Physiology).
ular Physiology) Volume 114, 21
5, 1983] has been reported. Examples of useful protein-producing cells cultured in a serum-free medium include monoclonal antibody-producing mouse hybridoma (Japanese Patent Application Laid-open No. 58-63385) and interferon-producing cells.
It is known that α-producing human Burkitt's tumor-derived Namalva cells are made serum-free by acclimatization (Tissue Culture, Vol. 9, p. 286, 1983). These are all examples of fused cells and so-called spontaneously producing cells. On the other hand, genetic recombination technology has made it possible to produce useful proteins in any type of cell. For example, production of interferon-β using L cells as hosts (Proc.

Natl, Acad, Sci, USA Vol. 79, p. 5166, 1982], interferon-γ using CHO cells as host [Journal of Interferon 6 Research (Journal of Interferon 6 Research)
4eron Re5earc-h) vol. 4, p. 461, 1984] and TPA production (JP-A-59-4232)
1) and production of human growth hormone using C127 cells as hosts (Proc.

Natl, Acad, Sci, USA vol. 80, 3
97, 1983], there are many examples of production of useful proteins using animal cells. However, serum is used for culturing these production strains created using genetic recombination technology, and as mentioned above, there are problems in terms of cost and purification, so culturing in serum-free media is desired. . However, after obtaining a transformed strain, it requires more time and effort to make it serum-free, and it is estimated that it may be difficult to make it serum-free. Culture has not been carried out.

"Problems to be Solved by the Present Invention" In the production of useful proteins using animal cells as hosts using genetic recombination technology, various attempts have been made to increase productivity. (MTX) resistance due to dihydrofolate reductase (d
h f r”) gene amplification method, it has been reported that 50 μg of the target protein can be produced per 106 cells (
In the production of substances using animal cells, it is equally important to select production cells that can be cultured without serum, rather than simply breeding to improve productivity.

Serum contains a wide variety of components, and even if the cells that are passaged in a serum medium are of the same strain, not all clones have the same requirements, and some cells may not have the same requirements. Even if a gene encoding a useful protein is introduced into a serum-containing cell as a host, if selection is performed in a serum medium, it is presumed that the auxotrophy will change during the selection and a serum auxotrophy will occur. In particular, dihydrofolate reductase (dhf) is treated with mesotroxate (MTX), which is currently often used as a breeding method for high-producing strains.
r) In the gene amplification method, the concentration of methotrexate is increased stepwise for selection, so there is a high probability that cells will become serum-requiring.

In fact, it was difficult to make a high-producing strain serum-free under conditions that would allow host cells to become serum-free after obtaining a high-producing strain.

"Means for Solving the Problems" In view of the above circumstances, the present inventors have conducted intensive studies to obtain cells that produce useful proteins and can be cultured without serum. As a result of searching for a basal medium suitable for addition of factors and selection, and selecting cells in a serum-free medium, it became clear that a production strain that could be cultured without serum could be obtained.

The growth factors used in the serum-free medium are not particularly limited, and depending on the cell type, insulin may be used alone, or transferrin, fibroblast growth factor, epidermal growth factor,
It is also possible to use a combination of platelet-derived growth factors and the like. In addition, the basal medium containing sugars, amino acids, vitamins, nucleic acids, etc. can be used alone, such as MEM medium, Dulbecco's modified MEM medium, 199 medium, RPM11640 medium, Ham's F12 medium, MCDB medium, etc., which are general mediums for animal cells. Alternatively, a mixed medium is used, but depending on the cell selection conditions, it is necessary to consider the basal medium. For example, dihydrofolate reductase (dhfr) can be
When performing gene amplification, αME, which does not contain nucleic acids, is generally used.
M medium is often used, but when selecting with a serum-free medium, good results can be obtained by using a basal medium that does not contain nucleic acids and has good cell proliferation. type of basal medium,
Regarding the composition [Mesofushi in Enzymology (
Method in Enzymology＾cad
emic press vol. 58, p. 62, 1979]
is described in detail.

Genes encoding proteins necessary for selecting transformed cells include the gene encoding Ecogpt derived from Escherichia coli (R, C, Mulligan + P, Berg,
+Proc.

Natl, Acad, Set, USA, vol. 78, p. 2072, 1981], bacterial transposons (
Tn) 5-derived Neo gene (P, J, 5other
r++ P, Berg, (Journal of Molecular and Applied Genetics (Jo)
urnal of Moleculer a-and A
pplied genetics) Volume 1, Page 327,
1982], herpesvirus-derived tk gene CM for thymidine kinase (tk)-deficient hosts, Wigl
er et al., Cell, 11i8.223, 1
[977], any other gene can be used as long as it is possible to select a transformed strain. Similarly, the gene encoding dihydrofolate reductase (dhfr) is mouse dhfr.
In addition to cDNA, genes derived from other animals or genes derived from chromosomes are acceptable.

Methods for introducing DNA into animal cells include the calcium phosphate method (CM Jigler et al., Ce1l, vol. 11, p. 223, 1977), the microinjection method (W, F
, Anderson et al., Proc. Natl., Ac.
ad, Sci, USA.

77, p. 5399, 1980], cell fusion method (W,
5choffner et al., Proc Natl., Ac.
ad, Sci, USA, vol. 77, p. 2163, 19
1980] etc. are used.

"Effects of the Invention" The serum-free cultureable protein-producing cells obtained by the present invention can be cultured in large quantities as shown in the examples, and like CHO cells, they can adhere and grow in normal serum media. It was found that cells that can be cultured in suspension can be obtained in large-scale culture because these cells grow in suspension, similar to leukocyte-derived cells.

Furthermore, according to the present invention, useful protein-producing cells that can be cultured without serum can be reliably obtained, and as shown in the examples, useful proteins in the culture supernatant can be obtained with high purity, which is advantageous in terms of industrial, cost, and purification. This provides an advantageous method.

"Example" Hereinafter, the present invention will be explained by giving examples.

Although CHO cells will be described as a specific example of the present invention, BHK is a cell that has the potential to be industrially useful.

There are many other cells such as Vero, CEM, Namalva, etc., and the invention is not limited to the illustrative cells. Furthermore, as examples of useful proteins, interferon-γ, tissue plasminogen activator, and lymphotoxin are described as examples, but of course they are representative examples, such as erythroboetin, interleukin-2, interfusilon-β, There is nothing wrong with stimulating factors or anything else.

Example 1: Obtaining interferon-T producing transformed strain Host cells were prepared using basal medium MCDB302/Dulbetsuko MEM
Insulin 5μg/1/1 mixed medium (MD medium)
CHO-K I acclimated to serum-free medium supplemented with m1
using cells. Vector psVe containing Ecogpt gene, dhfr gene, and interferon-T gene
dhfr-r2 (patent application Sho 6O-260450), D
100 μg of NA was transfected into 1×10′ CHO cells by the calcium phosphate method. Cell number 1 × 10 in MD medium containing 5 μg/m insulin
S cells/ml, 25 μg/m j! of mycophenolic acid, 25 (Jug/mA! of xanthine, 0.
Aminobutyrin at 1 μg/mj2, 5 μg/m I
Add t thymidine and 25 μg/m J adenine and place in a Corning 96We 11 multiplate (
No. 25860) 100. c+j! /Wel1, and cultured in an incubator at 37° C. and 5% CO. The interferon-γ activity of the wells in which the cells were grown was measured, and interferon 1500 u/mj, which can be cultured without serum! A producing transformed strain was obtained.

Assay for interferon-T activity was determined by inhibition of the cytopathic effect of Sindbis virus on FL cells (
(see JP-A-59-119648) and a standard interferon sample (Gg23901-530).

Example 2: Obtaining a serum-free culturable interferon-T high producing strain The serum-free culturable transformant obtained in Example 1 was cultured in MD medium containing 500 nM methotrexate (MTX) (+5 μg/m insulin). 1), the number of cells is 10
''~104 cells/ml, dispense 100μj!/We11 into 96Well+multiplate, 37℃, 5%C
Cultured in an Ot incubator. MT after 3-4 days
100 μj of serum-free medium containing 500 nM of X! /Wel
10 minutes until the MTX-resistant strain grows every 3 days.
The medium was replaced at 0 μm/well. The interferon production amount of the MTX-resistant strains that appeared was measured, and further MTX
Resistant strains were obtained in the same manner as above at a concentration of 1500 nM.

As a result of interferon-γ assay, interferon-γ can be cultured without serum at 300,000 U/m! The above production strains C
HO-590 strain was obtained (Figure 1).

Example 3: Purity of interferon-γ in culture supernatant Interferon-γ producing strain CHO obtained in Example 2
-590 was cultured in serum and serum-free medium, interferon-γ and protein were measured on the 5th day of culture to determine specific activity, and the culture supernatant was subjected to SDS polyacrylamide electrophoresis. A Biorad protein assay kit was used for protein quantification.

Example 4: Mass culture interferon producing cells CHO-5 shown in Example 2
Ninety strains were cultured in mini jars. Seed cells at 37°C, 5
% CO in an incubator. RDF containing insulin 5μg/m1 in a fi3J mini jar
A medium (RPM11640/Dulbetzko's modified MEM/Ham's F+t=2/1/1) was dispensed into 11 portions, and seed cells were inoculated.

CHO-590 strain can be cultured in suspension at a temperature of 37°C.
Medium perfusion culture was performed at pH 7.0. The medium perfusion rate was increased as the cell density increased (Figure 2).

Example 5: Obtaining a strain with high lymphotoxin production that can be cultured without serum
-23637) DNA GCHO cells were transfected by the calcium phosphate method. Transformants were selected in the same manner as in Example 1 using a serum-free medium. Cells grew W
measuring lymphotoxin activity in the culture supernatant of ELL,
A lymphotoxin-producing transformed strain was obtained.

The transformed strain was subjected to methotrexate (MTX) selection in the same manner as in Example 2 in a serum-free medium. By further selecting the amplified strain obtained with MTX 250 nM using MTXIOoonM, lymphotoxin 180,000 u/m j! A cell line that can be cultured without serum was obtained.

Example 6: Obtaining a high-producing tissue plasminogen activator (TPA) strain that can be cultured without serum. Vector pSVePA-3 containing Ecogpt gene, dhfr gene, and TPA gene (Patent application No. 61-9748)
1) DNA was transfected using the calcium diphosphate method in CHO cells. Selection was made in a serum-free medium in the same manner as in Example 1, and TPA-producing transformants were obtained by measuring TPA activity.

The transformed strain was treated with MTX in a serum-free medium in the same manner as in Example 2.
Made a choice. MTX? n degree 250 nM.

TP was amplified three times at 750 nM and 2000 nM.
A serum-free culture producing A1400 u/ml was obtained.

[Brief explanation of the drawing]

Figure 1 shows the growth of CHO-590 strain in serum-free medium and I
FN-r production curve. FIG. 2 is a production curve of IFN-γ in mini jar culture using CHO-590 strain.

Claims (1)

[Claims] 1. A transformed cell into which a gene encoding a useful protein has been introduced and which can be subcultured in a serum-free medium. 2. The cell according to claim 1, wherein the host cell is a cell that can be subcultured in a serum-free medium. 3. The cell according to claim 1 or 2, wherein the host cell is a CHO cell, a Hela cell, or a cell derived from these cells. 4. The cell according to claim 1, 2, or 3, wherein the useful protein is interferon-γ, tissue plasminogen activator, or lymphotoxin. 5. A method for breeding transformed cells that can be subcultured in a serum-free medium by selecting cells into which a gene encoding a useful protein has been introduced in a serum-free medium. 6. The method according to claim 5, wherein the host cell is a cell that can be subcultured in a serum-free medium. 7. The method according to claim 5 or 6, wherein the host cell is a CHO cell, a Hela cell, or a cell derived from these cells. 8. The method according to claim 5, 6, or 7, wherein the useful protein is interferon-γ, tissue plasminogen activator, or lymphotoxin. 9. A method of producing a useful protein by culturing in a serum-free medium a transformed cell into which a gene encoding a useful protein has been introduced and which can be subcultured in a serum-free medium. 10. The method according to claim 9, wherein the host cells are cells that can be subcultured in a serum-free medium. 11. The method according to claim 9 or 10, wherein the host cell is a CHO cell, a Hela cell, or a cell derived from these cells. 12. Claims 9, 10, or 11, wherein the useful protein is interferon-γ, tissue plasminogen activator, or lymphotoxin.
The method described in section.