JP3523293B2 - Method for enhancing interferon production - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for enhancing the production of interferon which is useful as an anticancer agent or antiviral agent.

[0002]

2. Description of the Related Art Interferons are a series of substances defined by animal cells as proteins having an antiviral action, and three types of interferons, α, β and γ, have been found. Interferon-α is found as an interferon secreted by leukocytes and lymphoblastoid cells, and it is known that interferon-α has many subtypes and consists of 165, 166, 172 amino acids ( Biomedica, 6 (14), 43-49
Pages (Vol. 1379-1385) (1991)). In addition to antiviral effects, interferon is known to have multiple effects such as antitumor effect and effect on immune system cells (Shigeru Kobayashi, Interferon Science Kodansha Scientific 1985, p. 44).

The method for producing interferon-α includes a method of inducing leukocytes and lymphoblastoid cells with viruses such as Sendai virus and Newcastle disease virus, production by genetically modified microorganisms, production by genetically modified animal cells and the like. Although known, production by cell lines such as lymphoblastoid cells contains many subtype components, and naturally-occurring interferon-α (hereinafter referred to as natural type)
It is considered to be the closest and safer to.

From the viewpoint of productivity, lymphoblastoid cells can be mentioned as the production cells of the animal cell culture method. As the lymphoblastoid cells, Namalwa cell line is particularly preferable.
The Namalwa cell line is a cell line derived from Burkitt lymphoma and is a cell line with high interferon production (S. Karger,
Basel, Develop. Biol. Standard., 38 , 343-348 (197
8)). A method of culturing this cell line and using Sendai virus as an inducer is used for mass production as an efficient interferon production method (Methods in Enzymolog
y, 119 , 35-38). Furthermore, a method for enhancing interferon production is known by treating cells in advance with a production enhancer such as butyric acid, dimethyl sulfoxide, 5-bromodeoxyuridine, etc. before inducing interferon-α with an inducer such as Sendai virus. (Journal
of Interferon Research, 2 ( 2), 261-270 (1982)).

[0005] The treatment temperature of cells with a drug used to enhance the production of interferon-α is 36 to 3
7 ° C. (Japanese Patent Laid-Open Nos. 54-20119 and 57-57)
No. 74093, Japanese Patent Publication No. 60-18398).
However, even with the above method, the production amount in the animal cell culture method is lower than that in the gene recombinant microorganism method. Therefore, development of a production method in which a higher titer of interferon-α is secreted per unit cell has been desired.

[0006]

The object of the present invention is to increase the interferon production.

[0007]

The present invention relates to a method for enhancing interferon production by culturing cells in a medium containing an interferon production enhancer at a temperature of 35 ° C. or lower.

The method for producing interferon includes the following methods.

The animal cells are cultivated at a temperature of 36 to 38 ° C.,
The cells may be diluted with a medium containing a production enhancer,
Alternatively, cells may be separated by centrifugation or ultrafiltration, and the separated cells may be suspended in a medium containing a production enhancer.
This is incubated at a temperature of 26-35 ° C for 24 hours or more. Incubation time is 24 hours to 6
0 hours is good, but around 48 hours is suitable. The concentration of the production enhancer in the medium varies depending on the type of the production enhancer, but in the case of butyric acid, 0.5-5 mM is optimal. Furthermore, after the animal cells are treated with the production enhancer, induction is performed with the interferon inducer. To induce interferon, the interferon inducer is added to the animal cell culture medium containing the production enhancer, or the cells are separated by centrifugation or ultrafiltration of the animal cell culture medium, and the separated cells are free from the production enhancer. A method of adding an interferon-inducing agent after suspending the medium is mentioned. And 26-35 ° C
The interferon is secreted by incubating at the temperature of 8 hours or more. The produced interferon can be separated from cells by centrifugation or filtration, recovered as a supernatant or a filtrate, and isolated by an ordinary method such as precipitation, extraction with an organic solvent, or column chromatography (for example, Methods in Enzymology, 119 ,
39-47, Acta microbiol. Acad. Sci. Hung., 28 , 263-
270 (1981)).

The animal cells used for interferon production in the present invention are not particularly limited as long as they produce interferon, but cells producing interferon-α are preferred. Examples of such cells include leukocytes, leukemia cells, lymphocytes, and lymphoblastoid cells, but NC-37, BALL-1, and Namalwa cells are particularly preferable.

For culturing animal cells, these cells are cultured in a plastic, glass or stainless steel container. In addition, culture using hollow fibers such as hollow fibers is also possible.

Any medium can be used as long as it is usually used for culturing animals, and a commonly used Eagle minimum essential medium, Ha
m F12 medium, Dulbecco's modified Eagle medium, RPM
The I-1640 medium can be exemplified, and a mixed medium thereof may be used. The medium is usually supplemented with 0.5 to 10% animal serum. Examples of the serum include fetal bovine serum, fetal bovine serum, adult bovine serum, horse serum, human serum and the like, but protein hydrolysates such as casein hydrolysates and beef extract hydrolysates to reduce the amount of serum added. It can also be added. Further, proteins such as bovine serum albumin, horse serum albumin and human serum albumin can be used instead of animal serum.

Examples of the interferon production enhancer include straight chain lower fatty acids such as acetic acid, propionic acid, butyric acid, valeric acid and hexanoic acid, but butyric acid is preferred.

The most preferred embodiment of the present invention is a method of culturing Namalwa cells in the presence of butyric acid and then using Sendai virus (Kantel strain) as an interferon inducer.

[0015]

INDUSTRIAL APPLICABILITY According to the production method of the present invention, interferon production by animal cells can be enhanced, and interferon can be produced industrially advantageously. In particular, in the production of natural interferon-α, the production of interferon can be enhanced without impairing its properties.

[0016]

EXAMPLES The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

Embodiment 1 1. Cells and Reagents <Cells> Namalwa cells <Medium> RPMI-1640 (bovine serum, RPMI-) containing 7% or 1% bovine serum (bovine within 1 year and 6 months old)
1640 medium: Gibco) <Reagent> Sodium butyrate (Wako Pure Chemical Industries) Sodium propionate (Nacalai)

2. Induction of interferon- (Experiment 1) RPMI-1 containing 7% bovine serum under the condition of temperature 37 ° C
Namalwa cells subcultured in 640 medium 3.90 x 10
⁶ cells / ml was diluted 2-fold with RPMI-1640 medium containing 1% bovine serum, and 500 ml of cell solution added with sodium butyrate solution to a butyric acid concentration of 2 mM was added at 47 ° C and 31 ° C respectively. Cultured for 5 hours. Next, 100 ml of each cell solution was transferred to a spinner flask, and 0.2 v / v% concentration of Sendai virus (3.2 HA unit (Haemagglutinating unit) / ml) was transferred to the spinner flask.
So that the temperature of each cell culture solution is 33
Interferon induction was performed at ° C. 23 from the start of induction
After a lapse of time, the cell culture solution was centrifuged to collect the supernatant. Further, the Sendai virus contained in the supernatant was acidified with hydrochloric acid to inactivate it, and then neutralized again with an aqueous sodium hydroxide solution. Interferon-α concentration of the inactivated and neutralized supernatant was determined by radioimmunoassay (RIA method).
It was measured at.

2. Induction of interferon- (Experiment 2) Namalwa cells 2.93x subcultured in the same manner as in Example 1
RPMI-1640 containing 10 ⁶ cells / ml in 1% bovine serum
Twenty-five ml of the cell solution, which was diluted 2-fold with the medium and further added with a sodium propionate solution so that the sodium propionate concentration was 10 mM, was cultured at 37 ° C. and 35 ° C. for 46.7 hours respectively. Then 10m from each cell
Transfer 1 liter each to a plastic tissue culture flask and add Sendai virus to this at a concentration of 0.5 v / v% (8 HA units (Haemagglutinating unit) / ml) to prepare a cell culture medium at a temperature of 31 ° C. Interferon induction was performed. 23.5 hours after the initiation of induction, the cell culture medium was centrifuged and the supernatant was collected. After inactivating the Sendai virus and neutralizing the sample by the method described in Example 1, the interferon-α concentration was measured by the RIA method.

3. Interferon-α quantitative radioimmunoassay was performed. The antibody beads on which the anti-α-IFN polyclonal antibody (first antibody) was immobilized were bound to α-IFN in the sample by an antigen-antibody reaction. Further ¹²⁵ I in to bind the labeled anti-IFN monoclonal antibody (secondary antibody) bound to the beads ¹²⁵
The radioactivity of I was measured with a γ counter. At the same time, the same operation was performed using the interferon-α standard solution, and a calibration curve was prepared from the obtained measured values to determine the interferon-α content in the sample. The IFNα kit manufactured by Dynabot was used for the first antibody and the second antibody. Interferon-α standard solution is Interferon international standard product J-501
An in-house standard product prepared based on the above was used.

4. Results Table 1 shows the interferon production promoting effect of the treatment temperature of the interferon production enhancer.

[Table 1] *: Ratio to the production amount at the production enhancer treatment temperature of 37 ° C. As can be seen from this table, when the cells were treated with the production enhancer (linear lower fatty acid), the interferon-α production was significantly increased when the cells were treated at a temperature of 37 ° C or lower than 35 ° C.

─────────────────────────────────────────────────── ─── Continuation of the front page (58) Fields surveyed (Int.Cl. ⁷ , DB name) C12P 21/00 BIOSIS / WPI (DIALOG)

Claims (6)

(57) [Claims] 1. An interferon production method in which cells having interferon-producing ability are cultured in the presence of an interferon production enhancer, and then induced by an interferon production inducer to produce interferon, in the presence of an interferon production enhancer. Substantially 26
A method for producing interferon, which is performed at a temperature in the range of to 35 ° C for at least 24 hours or more . 2. Culturing in the presence of a production enhancer is at least
The manufacture of interferon of claim 1 wherein from about 48 hours
Law. 3. Culturing and induction in the presence of a production enhancer is carried out.
Characterized by a temperature in the range of qualitatively 26 ° C. ~ 35 ° C.
The manufacture of interferon of claim 1 or claim 2, wherein
Law. 4. The method for producing interferon according to claim 1, 2 or 3, wherein the production enhancer is a linear lower saturated carboxylic acid. 5. The method for producing interferon according to claim 4, wherein the production enhancer is butyric acid. 6. The method for producing interferon according to claim 1, 2, 3, 4, or 5, wherein the interferon is interferon α.