KR930006117B1 - Medium for animal cell culture - Google Patents

Abstract

The high concentration medium, e.g. MBRI 40-01, MBRI 40-02 and MBRI 40-03, for animal cell culture contains at least one or more of amino acids (I), vitamins (II), gultathione and glucose. Pref. the (I) is arginine, glutamine, isoleucine, proline, tyrosine etc., and the (II) is biotin, inositol, nicotine amide, vit. B12, riboflavin, etc.. The animal cell is cultured by innoculation in the medium contg. 2500 mg/l glucose, 750 mg/l glutamine, 1 % calf propagule serum etc. and fermenting under the air condition contg. 5 % CO2 gas at 37 deg.C.

Description

High concentration medium and culture process for animal cell culture

Figure 1 is a comparison of the growth curve comparing the proliferation of the cell fusion line 2c3.1 cells cultured in the high concentration medium group MBRI 40-01 and 40-02 enriched with all the nutrients of the conventional RPMI 1640 medium and RPMI 1640 medium.

Figure 2 is a comparison of the production curve of a single-cell antibody against hepatitis surface antigen produced by cell fusion cells cultured in a high concentration medium MBRI 40-01 enriched with all the nutritional components of RPMI 1640 medium and RPMI 1640 medium.

Figure 3 is a comparison of the growth curve comparing the proliferation of human lymphoma cells cultured in the high concentration medium group MBRI 40-01 and 40-02 enriched with all the nutrients of the conventional RPMI 1640 medium and RPMI 1640 medium.

Figure 4 is a comparison of the growth curve comparing the proliferation of human myeloma cells cultured in high concentration medium group MBRI 40-01 and 40-02 enriched with all the nutrients of the conventional RPMI 1640 medium and RPMI 1640 medium.

Figure 5 is a process chart showing the curve of the residual glucose concentration in the fed-batch cultivation process using high concentration medium MBRI 40-02 and 40-03 and the addition method of fed-batch high concentration medium based on the same.

6 is a cell proliferation curve of the cell fusion cell during the fed-batch culture process using high concentration medium MBRI 40-02 and 40-03.

FIG. 7 is a fish curve diagram of a single cell antibody against hepatitis surface antigen produced by cell fusion strains during the fed-batch culture of cell fusion strains using high concentration medium MBRI 40-02 and 40-03.

The present invention relates to a highly enriched nutrient reinforcing medium and a cell culture process that enables high-density cell culture to be close to the cell concentration in vivo, based on RPMI 1640 medium, which has traditionally been used for in vitro animal cell culture. . In particular, the high-density culture medium of the present invention overcomes the proliferation limit in in vitro culture of mammalian cells, and allows the growth of up to 5 times the proliferation limit.

In recent 5 years, as the development of the production of various drugs and new substances using mammalian cells has rapidly increased, the cells are classified into immobilization techniques or perfision cultures as the most suitable process for industrialization of products. High concentration culture technology of mammalian cells is rapidly being introduced. However, these techniques also have several limitations because they realize the idea of achieving high concentration culture by bypassing rather than solving complex problems in vitro culture. Biotechnol. 5,230, 1987 / Biotechnol.Bioeng. 28,646, 1986 / In Vitro 22 (10), 589, 1986 / J.Immunol.Method 86, 61, 1986 / Appl.Microbiol.Biotechnol.26,495, 1987 / European Patent No. EPO 205 790, 1986 / Commercial Production of Monoclonal Antibodies 119, 1986).

For example, in the case of immobilization methods (hollow fiber reactors, ceramic bioreactors and various immobilizing matrices), it is difficult to oversize the processes necessary for industrialization due to their dynamic characteristics, and in the case of perfusion culture or continuous culture method, the use of expensive medium or power This is inevitable and its improvement is not easy. On the other hand, the cell suspension culture method is already being used in practice since a lot of research on industrialization, and since it does not have the problems described above, in principle, it can be said that it is relatively suitable for industrialization. (Enzyme Microb. Technol. 9,607, 1987 / Biotechnol. Bioeng. 32,993, 1988). In view of this, the conventional in-situ culture technology development adds a specific energy source, amino acid, vitamin, etc. at a high concentration, thereby reducing the growth limit concentration of (1-3) × 10 ⁶ cell / ml of the in vitro batch culture of animal cells. Although this method was intended to overcome some of the effects of prolonging the survival of animal cells, it was not effective in overcoming the limit of cell proliferation. PCT Patent WO 87/00195, 1987 / Biotechnol. Lett. 9,691, 1987 / WO 90/340 (PCT / us, 89/03986).

The reason why these conventional methods have not succeeded in overcoming the limitation of cell proliferation is that they do not recognize the correlation between the nutrients and the fact that the nutrient utilization ability of the cells that grow in vitro is extremely inefficient. To do so requires a high concentration culture medium with all the nutrients in a balanced and fortified manner. In principle, complex interactions occur between the nutrients required by a cell, and thus, the rate of proliferation of the cell depends on the first consumed component. However, the media that have been used for cell culture so far have insufficient concentrations of media components to meet the nutrients required by the cells to overcome and propagate the limit growth level of in vitro cell culture. (Biotechnol. Bioeng. 36, 717 , 1990). Therefore, it can be said that only high concentrations of supply throughout the nutrient can solve this problem (Cell Culture Methods for Molecular and Cell Biology 1, 3, 1984).

The present invention relates to a suspension cell culture method for developing a high concentration medium in which the conventional medium for cultivating animal cells is balanced in general, and achieving high concentration cell proliferation using the newly developed medium. That is, the present invention, as shown in Table 1 below, the concentration of each inorganic salt except for Nacl in the conventional RPMI 1640 medium (J. Am. Med. Assoc. 199, 519, 1967), all amino acids, vitamins, glutathione Development of a series of high-density medium groups (MBRI 40-01, MBRI 40-02 and 40-03 high-density medium), 5 times, 10 times, 50 times strengthened, and 2.5 times stronger glucose, and the high concentration suspension cell culture process using the same will be. Nacl concentration was adjusted to osmotic pressure (Osmolality) 270 ~ 320 Osm / kg, water. MBRI 40-02 and 40-03 medium was also used to adjust the osmolality in water 250 ~ 320mm Osm / kg, by adjusting the concentration of Nacl below 2g / ℓ.

TABLE 1a

Concentrations in MBRI 40-01, 40-02, 40-03 and RPMI 1640 Medium

TABLE 1b

The present invention enables high concentration culture by using a high concentration medium, thereby providing high added value to the production of various medicines and bioactive substances produced by animal cells. In addition, the present invention enables the production of high concentrations of the materials, enabling the miniaturization of the conventional cell culture process, the simplification and miniaturization of the purification process, thereby minimizing the problems derived from the enlargement step of the process required for industrialization. The present invention is designed to eliminate the use of a high-concentration culture fermenter or device other than using a high-concentration medium, compared to the conventional cell culture technology, reducing the burden of investment and maintenance for the device, making the process easy to enlarge will be. The main factors contributing to the development of high-density medium were closely related to each other's molarity ratio, concentration of each medium component in the blood, cell growth inhibition concentration of each nutrient, and so on. Factors such as the percentage of nutrients and inorganic salts present, and the osmotic pressure of the medium (Textbook of Medicine, 1949, 1979).

In the present invention, non-adherent mammalian cells such as mouse fusion cell 2c3.1, human lymphoma cells, human myeloma cells, and the like were suspended and cultured using this medium, and the cell concentration was about 1 × 10 ^7. High concentration cultures that proliferate to cell / ml could be achieved. During the culture, each cell line showed high growth by using essential nutrients such as glucose and amino acids and vitamins in the medium which are about 2.5 times concentrated in the medium and 5 times concentrated in the medium.

As a result of the high concentration culture, the cell fusion line 2c3.1 produced single-cell antibody against hepatitis surface antigen at a concentration of 6 to 8 times higher than that produced by conventional in vitro culture. In the case of the fed-batch culture of high concentration medium, the cell concentration is maintained at a high concentration of (0.7 to 1.0) x 10 ⁷ cells / ml for 2,500 hours, and it is possible to continuously produce more than 25-fold single cell antibody against the surface of hepatitis. It was.

In order to compare the results of the high concentration culture, each cell line was inoculated at 1 × 10 ⁵ cell / ml and 1 × 10 ⁶ cell / ml, and MBRI 40-01 or MBRI containing 10% fetal calf serum in 100 ml each. Inoculated at 40-02, a high concentration medium and a conventional RPMI 1640 medium, the culture medium was stirred at 50 rpm in 37% of 5% CO ₂ atmosphere, and spinner flask culture was performed.

Example 1

[High Concentration Culture of Cell Fusion Line 2c3.1]

Cultured cell fusion cells were made by fusion of splenic cells of Balb c mice immunized with CRL 1580 myeloma cells (P3 × 63.Ag.8.653) and purified human hepatitis surface antigen (200 ㎍ / ml, Green Cross). It has been deposited with the Korea Microbiological Conservation Center, affiliated with the Korean spawn association, under the Budapest Treaty (KCCM 10003). Culture of the cells was usually carried out in RPMI 1640 medium containing 10% fetal calf serum and performed in an atmosphere containing 5% CO ₂ at 37 ° C. This cell fusion line is a cell line that continuously and stably produces a single cell group antibody of IgGl subtype against hepatitis surface antigen.

In order to compare the results of the high concentration culture, the cell fusion strains were inoculated at a concentration of 1 × 10.⁵cells / ml and 1 × 10⁶MBRI 40-01, 40-02 high concentration medium containing 10% fetal bovine serum in 100 ml each Inoculated in RPMI 1640 medium, 37% 5% CO₂In a containing atmosphere, spinner flask culture was performed with stirring at 50 rpm. The concentration of single-cell antibody produced in the medium was quantitatively analyzed by the following enzyme immunoassay.

Standard single-cell antibodies against hepatitis surface antigen (anti-HBs) were placed in polystyrene wells adsorbed with hepatitis surface antigen (HBsAg) and incubated at 37 ° C. for 2 hours. Standard antibodies purified by affinity purification were diluted with 0.15 M phosphate complete solution (pH 7.2) containing 5% bovine serum albumin to prepare a standard solution of 4.5-300 IU / ml. After incubation, each polystyrene well was added three times with 0.1 ml of 0.1% phosphate-polysorbate-20 buffer solution and washed three times. Then, 100 ng of hepatitis surface antigen-horseradish peroxidase conjugate diluted with 0.1 ml of normal phosphate. Was added and incubated at 37 ° C. for one hour. 0.1 ml of the substrate solution was added to each well, and the absorbance was measured at 405 nm using a spectrometer to quantitatively analyze the antibody. The standard antibody used was the supernatant obtained by mass culturing cell fusion strain 2c3.1 by affinity purification.

Cell fusion strains were inoculated with conventional RPMI 1640 medium and MBRI 40-01 medium at an initial concentration of 1 × 10 ⁶ cells / ml, followed by spinner culture. As shown in FIG. 1, RPMI 1640 medium After 22 hours of cultivation, the cell proliferation was rapidly increased to 2.3 × 10 ⁶ cells / ml, which is a normal cell proliferation limit, while the viable cell concentration rapidly decreased, whereas cultured in the medium of MBRI 40-01 high concentration 9.3. Proliferation was shown up to a concentration of 10 ⁶ cell / ml. Also, when inoculated at 1.5 × 10 ⁶ cells / ml, 10 times lower than 1 × 10 ⁶ cells / ml, as shown in FIG. 1, the original 80 × 8.0 × 10 ⁶ cells was observed. Proliferation to / ml gave a result corresponding thereto. In addition, as a result of culturing in MBRI 40-02 high concentration medium, the cell proliferation was increased up to the concentration of 1.2 × 10 ⁶ cells / ml, and the cell proliferation was increased by about 30% than in MBRI 40-01 high concentration medium.

As shown in Table 1, the high concentration medium MBRI 40-02 used in this example is a medium in which all nutrients except inorganic salts in MBRI 40-01 medium are doubled.

As a result of achieving a high concentration culture using a high concentration medium, the amount of single-cell antibody produced was also 6 to 8 times higher than the production by the conventional culture method as shown in FIG. When the cells were inoculated at 1 × 10 ⁶ cells / ml, after 118 hours of incubation, 340 µg / ml of antibody was produced, which was 6 times higher than 56 µg / ml obtained when cultured with conventional RPMI 1640 medium. High concentration. When the inoculation amount of the cells was about 1 × 10 ⁵ cells / ml, the culture yield of 450 μg / ml, which was eight times higher than that of the conventional medium, was observed after 161 hours of culture. In addition, antibody production in MBRI 40-02 high concentration medium was slightly higher than or equal to that in MBRI 40-01 high concentration medium.

As a result, the high-concentration medium developed in the present invention proved that the cell fusion strain contributed to a significant increase in the productivity of the single-cell antibody by providing a balanced supply of various nutrients required for high concentration growth above the normal growth limit.

Example 2

[Batch high concentration culture of human lymphoma cells]

Human acute lymphoblastic leukemia (CCRF-CEM; ATCC CCL 119) was usually placed in RPMI 1640 medium containing 10% fetal bovine serum and incubated at 37 ° C. in an atmosphere containing 5% CO ₂ . In order to compare the results of the high concentration culture, the cell line was inoculated concentration (1.3-1.5) x 10 ⁶ cells / ml, and a series of MBRI 40-01 and MBRI 40-02 each containing 10% fetal bovine serum in 100 ml. The medium was inoculated in a high concentration medium and RPMI 1640 medium, and maintained at 37 ° C. in a 5% CO ₂ atmosphere, and spinner culture was carried out with stirring at 50 rpm.

As shown in FIG. 3, the results of the high concentration culture were increased in 3.9 × 10 ⁷ cells / ml after 42 hours of cultivation in the conventional PRMI 1640 medium. Diesel oil cultured in MBRI 40-01 and MBRI 40-02 medium, respectively, after 96 hours of incubation, proliferated at high concentrations of 8.1 × 10 ⁶ cells / ml and 8.9 × 10 ⁶ cells / ml, respectively, Not only did they proliferate until doubled, but also maintained the activity of the cells for more than 50 hours, thereby demonstrating excellent culture capacity of the high concentration medium.

Example 3

[Batch Type High Concentration Culture of Human Myeloma Cells]

Human myeloma cells (RPM 8226; ATCC CCL 155) were usually placed in RPMI 1640 medium containing 10% fetal bovine serum and incubated at 37 ° C. in an atmosphere containing 5% CO ₂ . This cell line was inoculated at a concentration of 1.3 × 10 ⁶ cells / ml and inoculated into a series of high concentration medium of MBRI 40-01 and MBRI 40-02 containing 10% fetal bovine serum and 100 RPMI 1640 medium, respectively. Spinner flask culture was performed by maintaining the culture temperature at 36 ° C. in a 5% CO ₂ atmosphere and stirring at 50 rpm.

As a result of the high concentration culture, as shown in FIG. 4, when cultured in a conventional RPMI 1640 medium, after 28 hours of incubation to 1.5 × 10 ⁶ cells / ml, the activity of the cells rapidly decreased, whereas the high concentration medium MBRI When cultured in 40-01 and MBRI 40-02 medium, the maximum proliferation of cells was improved by 2.5 times after 76 hours of cultivation, respectively, up to 3.8 × 10 ⁶ cells / ml and 3.9 × 10 ⁶ cells / ml. In addition, the cell activity could be maintained for more than 50 hours.

Example 4

[Federated High Concentration Culture of Cell Fusion]

MBC 40-02 containing 10% fetal bovine serum in 100 ml at 1 × 10 ⁶ cells / ml of inoculation concentration, as in batch high concentration culture, 2c3.1 cell fusion strains producing single cell antibody against hepatitis surface antigen Inoculated in a high concentration medium, fed-batch culture was carried out with stirring at 50rpm in an atmosphere containing 5% CO ₂ at 37 ℃. In the fed-batch culture, the fed-batch culture medium containing 10% fetal calf serum, glucose, and glutamine (2.5 g / l, 750 mg / l, respectively) was added to MBRI 40-03 in Table 1 so that 10% of the total culture was used. Based on maintaining the residual glucose concentration at 1 g / l, it was carried out for 2,500 hours in a continuous addition mode, as shown in FIG. The arrow in the figure shows the timing of addition of the fed-batch culture medium.

As a result, as shown in FIG. 6, the cell concentration was maintained at a high concentration of at least 7 × 10 ⁶ cells / ml and at least 1 × 10 ⁷ cells / ml for at least 2,500 hours, and the amount of antibody produced was as shown in FIG. The fed-batch high concentration culture method was developed to continuously produce 1.0 g / l or more single-cell antibody at a high concentration of 2000 hours or more.

As a result, the high-concentration medium developed in the present invention proved that the cell fusion strain contributes to a significant increase in the productivity of the single-cell antibody by providing a balanced supply of various nutrients required for high concentration growth above the normal growth limit.

In summary, all of the above results are shown in Table 2. As a result of batch or fed-batch culture of cell fusion 2c3.1, human lymphoma and myeloma cells with a high-concentration medium prepared in the present invention, the proliferation of each cell line was compared with that of RPMI 1640, the highest cell proliferation concentration in batch culture. , 2.1-5.2 fold improvement, and single-cell antibody against hepatitis surface antigen produced by cell fusion strains was 6-8 fold higher than control culture. In addition, the cell line fed-batch culture was able to produce a high concentration of 3.0-5.2 times the cell growth for 2500 hours compared to the control culture, it was possible to produce a 27-fold single-cell antibody compared to the batch control culture.

TABLE 2

Always comparison of cell proliferation and production of high concentration cultures of various animal cell lines

Claims (4)

A medium MBRI 40-01 for animal cell culture containing RPMI 1640 medium containing amino acids, vitamins, glutathione and glucose in the same composition as the medium components of the medium name MBRI 40-01 shown in Table 1. Medium of the culture name MBRI 40-03 of Table 1, a medium for animal cell culture further comprising 1% fetal calf serum (W / V) with a glucose concentration of 2500 mg / L, glutamine concentration of 750 mg / L. Animal cells are inoculated in a high concentration medium MBRI 40-01 for culturing animal cells, and the culture of animal cells is carried out by continuously adding the medium of claim 2 to 10% of the total culture while stirring in a medium containing 5% CO ₂ at 37 ° C. . MBRI 40-02, an animal cell culture medium containing amino acids, vitamins, glutathione and glucose in the same composition as the media components of the medium name MBRI 40-02 shown in Table 1. Animal cells are inoculated in a high concentration medium MBRI 40-02 for culturing animal cells, and the culture of animal cells is carried out by continuously adding the medium of claim 2 to 10% of the total culture solution while stirring in an atmosphere containing 5% CO ₂ at 37 ° C. .