JPH05207874A - Serumless culture medium - Google Patents

Abstract

PURPOSE:To provide a production method for physiologically active substances using a serumless medium. CONSTITUTION:The objective serumless culture medium containing lambda- carrageenan. Using this medium, physiologically active substance-producing cells are cultured to produce physiologically active substances such as tissue-type plasminogen activating factor or monoclonal antibody. This serumless medium, which contains no serum or serum albumin, is inexpensive and stable. With this medium, even when adhesive cells fairly hard to culture are used, debonding of e.g. a microcarrier for the cells from an adherend wall can be suppressed, thus obtaining desired physiologically active substances at amounts equal to or more than those in the case with serum medium.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a serum-free medium used for producing various physiologically active substances by culturing animal cells, and a method for producing physiologically active substances using the medium.

[0002]

2. Description of the Related Art In recent years, with the rapid progress of genetic engineering technology and cell fusion technology, it has become possible to produce various useful physiologically active substances by animal cell culture. Conventionally, as a medium for animal cells, a medium to which a large amount of mammalian serum such as fetal bovine serum or calf serum has been added has been used irrespective of the cell proliferation process and the physiologically active substance production process.

However, these sera are very expensive, their prices fluctuate greatly, and because they are unstable liquid components, they must be frozen and stored, and it is difficult to obtain a large amount of them. , There is a problem in terms of mass supply. Further, since there are lot-to-lot differences in serum, the amount of accumulated physiologically active substance in the culture solution fluctuates, which hinders stable production of the physiologically active substance. Moreover, since serum contains many kinds of heterologous proteins, it is very complicated and difficult to remove these serum-derived substances from the culture solution.
It has also been an obstacle in the separation and purification of the target physiologically active substance.

Recently, a serum-free medium containing serum albumin instead of serum has been developed. However, it is necessary to add a large amount of this serum albumin, it is still expensive, there is a lot-to-lot difference like serum, and it is difficult to remove serum albumin when separating and purifying the desired physiologically active substance. However, most of the problems have not been solved yet.

Further, to date, serum-free medium containing no serum albumin has been developed and marketed centering on floating cells that are relatively easy to culture (Ono and Murakami, ed., Serum-free cell culture manual, Kodansha). , P31-39, 198
However, there are many problems for industrial use, such as being substantially unusable for adherent cells that are difficult to culture, having a short storage period, and being very expensive. Further, in the case of adherent cells, if the cells are grown in an appropriate growth medium such as serum medium and then switched to serum-free medium, the cells detach from the adherent wall, and there is a problem that the cells cannot be cultured for a long time. In particular, in the case of stirring culture such as a jar fermenter using a microcarrier for the purpose of scale-up, the cells tend to be more easily detached by the stirring shearing force.

From the above background, if it is possible to cultivate in a medium containing no serum or serum albumin in the production process of a physiologically active substance, regardless of adherent cells or floating cells,
It is possible to solve the problems of fluctuations in the production amount of physiologically active substances due to the difference between lots of serum and the removal of impurities in separation and purification. Furthermore, if the substance that replaces serum or serum albumin is inexpensive and stable, it becomes possible to greatly improve the problems in terms of economics of the medium and large-scale supply. Further, if the addition of these inexpensive substances can produce a physiologically active substance in an amount equal to or more than that in the serum medium, industrial mass production of the physiologically active substance becomes possible.

[0007]

An object of the present invention is to provide a medium for producing a physiologically active substance which does not contain serum or serum albumin. Another object of the present invention is to provide an inexpensive and stable serum-free medium. Still another object of the present invention is to provide a serum-free medium that suppresses detachment of cells from the adhesion wall even when adherent cells are used. Still another object of the present invention is to provide a serum-free medium capable of producing a physiologically active substance in an amount equal to or higher than that of the serum-free medium for a long period of time. Still another object of the present invention is to
It is intended to provide a method for producing a physiologically active substance using a serum-free medium.

[0008]

Means for Solving the Problems As a result of intensive studies for solving the above-mentioned problems, the present inventors have found that the process of producing a physiologically active substance does not include serum that is normally used in animal cells. By adding λ-carrageenan to the basal medium, it was found that, even when adherent cells are used, it is possible to produce a physiologically active substance in an amount equal to or greater than that of the serum medium over a long period of time, and the present invention was completed. Came to let me.

That is, the present invention provides a serum-free medium characterized by containing λ-carrageenan, and a method for culturing physiologically active substance-producing cells in the serum-free medium to produce a physiologically active substance. is there.

As the basal medium used in the serum-free medium of the present invention, any conventionally known serum-free basal medium for culturing animal cells can be used. Examples include Eagle's minimum essential medium, Dulbecco's modified Eagle medium, RPMI-1640 medium, Ham's F-12 medium and the like. It is also possible to mix a plurality of these basic media at an appropriate ratio or to add insulin, transferrin or the like for use.

Carrage used in the serum-free medium of the present invention
Nan is a kind of agar contained in red algae, and is a kind of acidic polysaccharide having agarobiose as a repeating unit and having a sulfate group bonded thereto. Although various types of carrageenan are known, λ type, which has almost no gelling ability, is used in the medium of the present invention. [lambda] -carrageenan is generally commercially available and can be easily obtained from Wako Pure Chemical Industries, Sigma Co., and the like.

In the present invention, when the amount of λ-carrageenan added to the medium is too much, in the case of adherent cells, the cells tend to be detached from the adherent wall.
When used in the range of 00 mg / l, favorable results are obtained.

As other components, sugar, amino acids, nucleic acid-related substances, vitamins, trace elements, protease inhibitors and the like may be added to the basal medium as needed. Examples of such components include the following. For example, glucose is generally used as a sugar source, and examples thereof include the case of adding galactose or the like for the purpose of reducing the amount of lactic acid accumulated in the medium during culture. In addition, for example, when culturing proline-requiring cells (such as Chinese Hamster-ovary cells) in the serum-free medium of the present invention using Dulbecco's modified Eagle medium (this medium does not contain proline) as a basic medium Then, an appropriate amount of L-proline is added. In addition, for example, a metallothionein promoter
When a physiologically active substance is produced by a recombinant cell transformed with the plasmid to which the target is connected, zinc, cadmium or a salt thereof is added to the serum-free medium of the present invention. Further, for example, in the serum-free medium of the present invention, if it is desired to produce more single chain of tissue-type plasmin-ogen activator than double chain, aprotinin,
A protease inhibitor such as ε-aminocaproic acid or p-aminomethylbenzoic acid is added. Further, for example, the case of adding butyric acid, propionic acid or a salt thereof to the serum-free medium of the present invention for the purpose of increasing the production amount of the physiologically active substance can be mentioned.

Needless to say, the serum-free medium of the present invention can be used even if serum or serum albumin is added. However, one of the objects of the present invention is to remove serum and serum albumin from the medium in the process of producing the physiologically active substance, so that when the physiologically active substance is recovered from the culture medium, it is disturbed by serum and serum albumin. If it occurs, the use of serum or serum albumin is not preferable.

The serum-free medium of the present invention is used for producing various physiologically active substances. Examples of such physiologically active substances include various enzymes (tissue-type plasminogen activator, etc.), various monoclonal antibodies (human monoclonal antibodies).
Null antibodies, mouse monoclonal antibodies, etc.), various lymphokines (interferon, etc.), various hormones (human growth hormone, etc.), and the like, which can be produced using the serum-free medium of the present invention.

As cells used for the production of such physiologically active substances, not only adherent cells which are relatively difficult to culture but also floating cells can be used. It is possible to produce physiologically active substances by culturing. Examples include fibroblasts, epithelial cells, lymphoid cells, transformed cells thereof, hybridomas and the like.

The method for culturing the physiologically active substance-producing cells for producing the physiologically active substance is not particularly limited, and a usual animal cell culture method can be used. For example, multi-well plates, petri dishes, tissue culture flasks, roller bottles, spinner flasks, jar fermenters, microcarriers, hollow fibers.
After inoculating an appropriate amount of cells into a growth medium (serum-free medium containing serum albumin or growth factors, or medium containing serum) suitable for the cells to be used, grow at a suitable temperature for a suitable time. , The medium for growth is removed, and the medium of the present invention is replaced to produce a physiologically active substance.

Further, for example, after the physiologically active substance is produced by the same method as described above, the culture solution is collected every several days and the operation of adding the fresh medium of the present invention is repeated to continue production. In addition, for example, after injecting an appropriate amount of cells into a growth medium suitable for the cells to be used and growing the cells at an appropriate temperature for an appropriate time, the medium of the present invention is continuously added, and at the same time, the growth medium is extracted. -Huth culture is performed to gradually remove serum albumin, growth factors, and serum, and finally completely remove them to produce a physiologically active substance.

[0019]

The present invention will be described in detail below with reference to examples. Example 1. Recombinant mouse C-127 cells, hT-382 strain, which are adherent cells and produce tissue-type plasmin-gen activator (hereinafter abbreviated as tPA) (Japanese Patent Laid-Open No. 62-
126978) was used. Microcarriers (Cytodex) in a Belco 500 ml spinner flask.
(1. Pharmacia) and 500 ml of Dulbecco's modified Eagle medium (growth medium) containing 10% of pre-inactivated fetal calf serum (hereinafter abbreviated as FCS) was charged. 7 × 10 ⁷ seed cells were seeded at 37 ° C. for 4
Culture was started under the condition of 0 rpm. On the third day of growth, the stirring was stopped, the microcarriers with attached cells were allowed to settle, and the culture medium was removed by suction. Fresh growth medium was added and the culture was continued again. On the 4th day of proliferation, the production medium was switched to produce tPA by the same procedure as above. As a medium for production, a Dulbecco's modified Eagle medium containing 20 μM zinc chloride and 10 mM ε-aminocaproic acid was used.
The medium to which various substances shown in (Table 1) were added was used.
After culturing for 2 days, tPA in the culture was quantified by the ELISA method. In addition, microscopic
The degree of cell detachment from the cells was observed. The above results are shown in Table 1.
The results are shown in (Table 1).

[0020]

[Table 1] (Note) Cell detachment (microscopic observation) The degree of cell detachment is higher in the order of +, ++, ++++, ++++.

Example 2. The same cells as in Example 1 were used. A microcarrier-in a Bellner spinner flask with an actual volume of 1 liter (total volume of about 1.5 liter) equipped with a stirring blade, in which a pH electrode, a DO electrode and a gas blowing tube were set.
(Cytodex 1, Pharmacia) and 5 g of Dulbecco modified method containing 10% of pre-inactivated FCS
1 liter of Glu's medium (medium for growth) was charged. 10 ⁸ seed cells above were seeded, 37 ° C., 30 rpm, DO = 1 to
Cultivation was started under the conditions of 2 ppm and pH = 7.0 to 7.2. On the 4th day of growth, the agitation was stopped, the microcarriers to which the cells were attached were sedimented, and the culture medium was removed by suction. Fresh growth medium was added and the culture was continued again. On the 5th day of growth, the production medium was changed to the production medium by the same operation as above
TPA was produced under the same conditions as above at 0 ° C. Insulin 5mg / l, transferrin 5mg in the production medium
/ L, zinc chloride 20 μM, ε-aminocaproic acid 10 m
Dulbecco's modified Eagle's medium containing M was added to Table 2 (Table 2).
The medium to which the various components shown in 1 above were added was used. After that, the medium was exchanged once every two days to produce tPA for 30 days.
The tPA was quantified by the ELISA method, and the average accumulated amount and the total accumulated amount were produced. On the 20th day of production, the degree of cell detachment from the microcarriers was observed with a microscope. The above results are shown in Table 2 (Table 2).

[0022]

[Table 2] (Abbreviation) λCA: λ-carrageenan Note: Cell detachment (microscopic observation) The degree of cell detachment is large in the order of +, ++, ++++, ++++.

Example 3. A hybridoma strain MP5121 (WO90 / 11350), which is a floating cell and produces a human monoclonal antibody (hereinafter abbreviated as IgM), was used. In a 25 cm ² tissue culture flask, add RPMI1
5 ml of a medium (growth medium) prepared by adding 10% of FCS to 640 medium was charged. 1 × 10 ⁵ seed cells of the above cells
The cells were inoculated so that the number of cells / ml was 1, and the culture was started in a carbon dioxide gas incubator at 37 ° C. On the third day of growth, the whole amount of the culture solution was extracted, the culture solution was removed by centrifugation, and the cells were separated. The cells were suspended in 5 ml of fresh growth medium, the cell suspension was returned to a 25 cm ² tissue culture flask, and the culture was continued. After culturing for 4 days in this way, the growth medium was removed by the same procedure, and the cells were suspended in the production medium at 1 × 10 ⁶ cells / ml, and 5 ml of the cell suspension was used for tissue culture. The flask was returned to the flask, and the production of IgM was started at 37 ° C. Insulin 10 mg / l, transferrin 10 mg / l
A medium in which various components shown in Table 3 (Table 3) were added to the RPMI1640 medium containing After that, the operation of recovering almost the entire amount of the production medium in the same manner as above and suspending the cells in 5 ml of the fresh production medium was repeated every day.
IgM was produced for 30 days. Ig in the recovered culture solution
M was quantified by the ELISA method and the total IgM production amount for 30 days was calculated. The results are shown in Table 3 (Table 3).

[0024]

[Table 3]

[0025]

EFFECTS OF THE INVENTION In the present invention, it is possible to provide a medium that does not contain serum or serum albumin regardless of adherent cells or floating cells. That is, as shown in the examples, when the adherent cells, which are relatively difficult to culture, are grown in an appropriate medium and then switched to the serum-free medium of the present invention,
It is effective in suppressing detachment of cells from the adhesion wall. Furthermore, when a physiologically active substance is produced in the medium of the present invention for a long period of time, it shows a total accumulated amount of physiologically active substances which is equal to or higher than that of the serum medium, and it is possible to inexpensively produce a large amount of physiologically active substances by animal cell culture. It is extremely effective.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location C12P 21/08 8214-4B // (C12N 9/72 C12R 1:91) (C12P 21/08 C12R 1:91)

Claims (5)

[Claims] 1. A serum-free medium comprising λ-carrageenan. 2. The concentration of λ-carrageenan is 10 to 100.
The serum-free medium according to claim 1, which is mg / l. 3. A method for producing a physiologically active substance by culturing the physiologically active substance-producing cell in the serum-free medium according to claim 1 or 2. 4. The method for producing a physiologically active substance according to claim 3, wherein the physiologically active substance is a tissue-type plasmin-ogen activator. 5. The method for producing a physiologically active substance according to claim 3, wherein the physiologically active substance is a monoclonal antibody.