JP3128882B2 - Method for producing interleukin-6 - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an interleukin-6.
And a method for mass-producing.

[0002]

2. Description of the Related Art Interleukin-6 (hereinafter referred to as IL-6)
Abbreviation) is a B lymphocyte differentiation factor, interferon β
₂ , 26K protein, a hybridoma / plasmacytoma growth factor, or a unified name of a cytokine called hepatocyte stimulating factor. IL-6 induces activated B cells to differentiate into antibody-producing cells. T
For cells, T cells stimulated with mitogen
Induces L-2 production and induces the IL-2 receptor in certain T cell lines and thymocytes. For hematopoietic cells, IL-6 synergistically induces the proliferation of hematopoietic stem cells in the presence of IL-3. Recently, it has been reported that IL-6 has an action as thrombopoietin. Thus, IL-6 has many physiological activities, and is expected to be applied as a medicine.

[0003]

At present, IL-6 is produced and provided as a recombinant. However, products produced by genetically modified Escherichia coli do not have sugar chains.
In addition, a product produced by a recombinant CHO cell may have a different sugar chain structure from a natural product. It is thought that the difference in the sugar chain structure affects the physiological activity and pharmacokinetics. In order to clinically apply properties such as the physiological activity and pharmacokinetics of natural IL-6, mass production of natural IL-6 is required. It is known that native IL-6 is produced, for example, by stimulating fibroblasts with certain cytokines. It has been reported that various stimuli as described below cause the production of IL-6, but they do not always show sufficient production for production on an industrial scale.

Conventionally, as a method for culturing adhesion-dependent cells such as normal diploid fibroblasts, a method using a roux bottle or a roller bottle is known. In this method, a large amount of adhesion-dependent cells are cultured. It is considered extremely difficult to do so. That is, in this method, cells grow only in a monolayer on the bottom of the roux bottle or on the inner surface of the roller bottle, so that a large number of bottles are required for mass culture. Also,
It is almost impossible to control the culture conditions such as the pH and the concentration of dissolved oxygen in the medium constantly. An object of the present invention is to provide a method for producing IL-6, which is capable of controlling a large amount of culture conditions constantly for clinical application.

[0005]

The above object is achieved by the present invention described below. That is, the present invention provides an interleukin characterized in that an IL-6-producing cell and an adhesion-dependent animal cell are cultured by adhesion to a microcarrier or a hollow fiber or immobilized to a microcapsule.
6 is a production method.

[0006] Native IL-6 is produced constantly by various cells or by various stimuli. Specifically, IL-6 is composed of floating cells represented by T cells, B cells, and monocyte macrophages, and adhesion-dependent cell lines represented by fibroblasts, osteosarcoma cells, lung cancer cells, and vascular endothelial cells. Produced by The present invention relates to the production of native IL-6 using adhesion-dependent animal cells. The present invention provides a method for producing adherent-dependent animal cells by adhesion culture on microcarriers or hollow fibers, or by immobilization culture in microcapsules, to produce IL-6 constantly without using an inducer, A method for producing IL-6, which comprises stimulating production with a -6 inducer.

The microcarrier used in the present invention is:
The matrix material is not particularly limited, and examples thereof include natural materials such as collagen, gelatin, cellulose, and cross-linked dextran, and materials made of synthetic resins such as polystyrene. These microcarriers can be charged or uncharged, but both can be used. Among the charged ones, a positively charged one is more preferable than a negatively charged one. Examples of the positively charged group include dimethylaminopropyl, dimethylaminoethyl, trimethylhydroxyaminopropyl and the like. Further, a matrix material coated with collagen, gelatin or the like can also be used. As a specific commercial product, “Cytodex-1” obtained by adding dimethylaminoethyl to cross-linked dextran
(Pharmacia) and "Cytodex-3" (Pharmacia), which is a crosslinked dextran coated with denatured collagen, but is not limited thereto. Cyt
When Odex-1 "(Pharmacia) is used, its concentration (amount used) is selected in the range of 0.05 to 1% relative to the culture solution, and preferably in the range of 0.2 to 0.7%. Is selected as appropriate.

The hollow fibers used in the present invention include those using modified cellulose and the like.
itafiber "(Amicon) and the like.
Microcapsules are formed by embedding cells inside using collagen or sodium alginate which forms a gel having water permeability (A. Klausner, Bio / technol., 1 , 736 (1.
983)).

To culture IL-6-producing cells and adhesion-dependent animal cells, seed cells are inoculated into microcarrier beads or hollow fibers, or gel-immobilized cells are prepared and cultured. The medium composition, serum concentration, and the like are appropriately selected according to the type of cells to be cultured, the cell concentration, and the like. During the culturing, the medium is replaced as appropriate, and the cells are cultured for several days to 20 days. Continue culturing until cells are almost confluent. It is desirable that the proliferated cells be incubated for several hours to several days with the addition of a low unit of cytocanin such as interferon and a negatively charged water-soluble polymer prior to treatment with the inducer. As interferon to be pretreated, interferon αs, interferon β, interferon γ and the like are used, and interferon αs and interferon β are particularly preferable. The concentration of interferon is usually 0.1 to 10,000 international units / ml, but more preferably 1 to 1000 international units / ml.

When cells that do not require an inducing agent such as vascular endothelial cells are used as cells, IL-6 can be produced constantly by merely culturing the cells. on the other hand,
When using IL-6-producing cells by stimulating an inducer such as fibroblasts, osteosarcoma cells, or lung cancer cells, the proliferating cells are treated with the inducer to produce IL-6.

Methods for treating cells with an inducer to produce IL-6 include synthetic RNA such as poly (I): poly (C) (hereinafter abbreviated as poly I / C) or natural RN.
A, etc., or IL-1, TNF, IFN-β
Such as cytokines, or growth factors such as PDGF,
Alternatively, a method of inducing with PMA, PHA, lipopolysaccharide, cholera toxin, or the like is used (Sc
issue, 235 , 731 (1987)). Among them, synthetic RNA or natural RNA is preferable, and it is particularly preferable to use poly I / C which is synthetic RNA. The conditions for inducing and stimulating poly I / C are not particularly limited.
g / ml, particularly preferably from 1 to 200 μg / m
1 is used. The poly I / C stimulation time is usually selected from several minutes to several tens of hours, but is preferably selected from 1 to 10 hours.

Further, after stimulating the cells with an inducer,
A method of enhancing production by treating cells with a metabolic inhibitor such as verapamil, cycloheximide, actinomycin D (J. Immunol., 144 , 424).
2-4248 (1990)).

[0013] The production cells are not particularly limited, but adhesion-dependent cells are selected from the characteristics of the present invention.
Particularly preferably, fibroblasts are used. Human IL-6
For the production of, human fibroblasts are selected. When human fibroblasts are normal diploids, 50
Although there is a limitation of about the generation, it can be used as a cell for industrial production by selecting culture conditions.

Further, the present invention provides that it is preferable to control the concentration of dissolved oxygen and the pH in the culture solution to a constant level during the culturing or the induction treatment of the IL-6 producing cells. When cells that produce IL-6 by stimulation of an inducer are used as the IL-6 producing cells, it is particularly preferable that the concentration of dissolved oxygen and the pH in the IL-6 production medium after growth are controlled to be constant. The dissolved oxygen concentration in the culture solution is always 20% to 80% with respect to the saturation solubility in air.
%, Particularly preferably in the range of 40% to 65%.
Similarly, it is preferable to control the pH in the range of 7.0 to 8.0. It is preferable that the concentration of dissolved oxygen and the pH of the present invention are constantly controlled within a specified concentration range.

[0015] The medium used in the present invention may be a usual one and is not particularly limited. Various commercially available mediums for culturing animal cells are generally suitably used, and the additives of these commercially available media are modified (the amount of the additives is slightly changed,
A medium in which certain additives have been removed, and in which certain additives have been added, etc.) is also preferably used. Among these, it is preferable to appropriately select a medium suitable for the cells and conditions.

[0016]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the invention is limited thereto.

Example 1 Crosslinked dextran microcarriers having 5% fetal bovine serum and diethylaminoethyl group ("Cytodex-
1 "(Pharmacia) Eagle ME containing 3 g / L
Approximately 2 × 10 4 human normal diploid fibroblasts in 2 L of M medium
^The cells were cultured in a spinner flask inoculated at a rate of ⁵ cells / ml at 37 ° C, pH 7.2, and 20% saturated oxygen concentration for 6 days with gentle stirring. On the way, the medium was changed on the first, third, and fifth days. The number of cells reached is 1.5 × 10 ⁶ cells / m
l.

Next, interferon-β (Toray Industries, Inc.)
(International natural interferon β, the same applies hereinafter) 100 international units / ml, exchanged for Eagle MEM medium containing carboxymethylcellulose, 37 ° C, pH 7.2, 20%
Incubated for 24 hours at saturated oxygen concentration. Next, after adding 10 μg / ml of poly I / C and incubating at 37 ° C. for 2 hours, the medium was replaced with an Eagle MEM medium, and further cultured at 37 ° C., pH 7.2, and 20% saturated oxygen concentration for 6 days. The amount of IL-6 finally produced depends on the amount of anti-IL-
6 antibodies (N. Ida et al., Biochem. Biophy).
s. Res. Commun. , 165 , 728-734
(1989)). The results indicate that the poly I / C was 0 μg / ml and the relative titer was 1.
00, when stimulated with 1 μg / ml, 1450,
It was 4460 when stimulated with 10 μg / ml.

Example 2 Crosslinked dextran microcarriers having 5% fetal bovine serum and diethylaminoethyl group ("Cytodex-
1 "(Pharmacia) Eagle ME containing 3 g / L
Human diploid cells were inoculated at a rate of about 2 × 10 ⁵ cells / ml in a spinner flask into 2 L of M medium at 37 ° C., pH 7.2 at 20% saturated oxygen concentration with gentle stirring.
Cultured for days. On the way, the medium was changed on the first, third, and fifth days. The number of cells reached was 1.5 × 10 ⁶ cells / ml.

Next, the medium was replaced with an Eagle MEM medium containing 100 units of interferon-β international unit / ml and carboxymethylcellulose at 37 ° C., pH 7.2, and 20%.
Incubated for 24 hours at saturated oxygen concentration. Next, after adding 10 μg / ml of poly I / C and incubating at 37 ° C. for 2 hours, the medium was replaced with an Eagle MEM medium, and further cultured at 37 ° C., 20% saturated oxygen concentration, pH 6.8 to 7.5 for 6 days. did. The amount of IL-6 finally produced is
Anti-IL-6 antibodies (N. Ida et al., Biochem. Bi).
ophys. Res. Commun. , 165 , 728
-734 (1989)). Relative IL-6 with pH 6.8 as 100
Was 208 at pH 7.2 and 218 at pH 7.5.

Example 3 Crosslinked dextran microcarriers having 5% fetal bovine serum and diethylaminoethyl group ("Cytodex-
1 "(Pharmacia) Eagle ME containing 3 g / L
Human diploid cells were inoculated at a rate of about 2 × 10 ⁵ cells / ml in a spinner flask into 2 L of M medium at 37 ° C., pH 7.2 at 20% saturated oxygen concentration with gentle stirring.
Cultured for days. On the way, the medium was changed on the first, third, and fifth days. The number of cells reached was 1.5 × 10 ⁶ cells / ml.

Next, the medium was replaced with an Eagle MEM medium containing 100 international units / ml of interferon-β and carboxymethylcellulose at 37 ° C., pH 7.2, and 20%.
Incubated for 24 hours at saturated oxygen concentration. Next, 10 μg / ml of poly I / C was added, and the mixture was incubated at 37 ° C. for 2 hours. Then, the medium was replaced with an Eagle MEM medium, and further cultured at 37 ° C., pH 7.2, and 5 to 50% saturated oxygen concentration for 6 days. The amount of IL-6 finally produced depends on the amount of anti-I
L-6 antibody (N. Ida et al., Biochem. Biop).
hys. Res. Commun. , 165 , 728-7
34 (1989)). 5
The relative titer of IL-6 production at 1% saturated oxygen concentration was 1
Assuming 00, 120% and 50% at 10% saturated oxygen concentration
The saturated oxygen concentration was 180.

Example 4 Crosslinked dextran microcarriers having 5% fetal bovine serum and diethylaminoethyl group ("Cytodex-
1 "(Pharmacia) Eagle ME containing 3 g / L
About 2 × 10 ⁵ human normal diploid cells / 2 L of M medium
The cells were cultured for 6 days at 37 ° C., pH 7.2, and 20% saturated oxygen concentration with gentle stirring in a spinner flask inoculated at a ratio of ml. On the way, the medium was changed on the first, third, and fifth days. The number of cells reached was about 1.5 × 10 ⁶ cells / ml.

Next, human interferon-β was reduced to 0,
1, 10, 100 international units / ml or human interferon-α (purchased from Genzyme) for 10
International unit / ml or human interferon-γ
Eagle MEM containing 10 international units / ml (purchased from Genzyme) and carboxymethylcellulose
The medium was replaced, and the mixture was incubated at 37 ° C., pH 7.2, and 20% saturated oxygen concentration for 24 hours. Next, after adding 10 μg / ml of poly I / C and incubating at 37 ° C. for 2 hours, the medium was exchanged with an Eagle MEM medium, and further, at 37 ° C.
The cells were cultured at pH 7.2 at 20% saturated oxygen concentration for 6 days. The amount of IL-6 finally produced depends on the amount of anti-IL-6 antibody (N.
Ida et al., Biochem. Biophys. Res.
Commun. , 165 , 728-734 (198
The measurement was performed by the enzyme antibody method using 9)). The relative titer of IL-6 production without interferon was 10
Assuming 0, interferon-β is 1, 10, 100
The relative titers of IL-6 when adding international units / ml are 200, 500 and 3000, respectively, and when adding 10 international units / ml of interferon-γ, 200 and adding 10 international units / ml of interferon-α. When 2
000.

[0025]

According to the present invention, natural IL-6 can be stably produced at a high yield. In addition, it is easy to control the culturing conditions at optimal values, and this is a culturing method suitable for scaling up to industrial production scale.

──────────────────────────────────────────────────続 き Continuation of the front page (56) References JP-A-1-308234 (JP, A) JP-A-62-100284 (JP, A) JP-A-59-146598 (JP, A) JP-A-60-1985 102187 (JP, A) JP-A-62-163687 (JP, A) JP-A-1-127037 (JP, A) JP-A-61-209586 (JP, A) JP-A-61-88893 (JP, A) Special Table Hei 5-502369 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12P 21/00-21/06 BIOSIS (DIALOG) WPI (DIALOG)

Claims (3)

(57) [Claims] 1. An interleukin-6-producing cell and an adhesion-dependent animal cell are adherently cultured on a microcarrier or hollow fiber, or immobilized on a microcapsule.
Cells until they are confluent.
When interleukin-6 is produced by treatment with a bulking agent
The concentration of dissolved oxygen in the culture solution after cell growth
Range from 20% to 80% of the saturation solubility.
A method for producing interleukin-6 , wherein the pH is maintained in the range of 7.0 to 8.0 . 2. The concentration of dissolved oxygen in a culture solution is determined by the
Maintained in the range of 40% to 65% of total solubility
The method for producing interleukin-6 according to claim 1,
Law. 3. After culturing the cells, the cells are treated with interferon
And then from synthetic or natural RNA.
4. The method according to claim 1, wherein the substance is treated with an inducer.
A method for producing interleukin-6.