JPS6135794A - Production of interferon-alpha - Google Patents

Abstract

PURPOSE:To increase the production of interferon-alpha (abbreviated as IFN-alpha) and to enable the mass production of IFN-alpha, by containing an IFN-alpha-producing cell with a lower alcohol, and inducing the cell. CONSTITUTION:An IFN-alpha-producing cell (e.g. cultured lymphoblastoid cell such as Namalva strain) is cultured in a medium to produce IFN-alpha. In the above process, the IFN-alpha-producing cell is made to contact with a lower alcohol (e.g. ethanol) prior to the induction. The contact is carried out preferably in a medium having the ultimate concentration of the lower alcohol of 0.1-3(V/V)%. The production of IFN-alpha can be increased, and a large amount of IFN-alpha can be produced by this process.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a method for producing interferon-α (hereinafter referred to as IFN-α). More specifically, the present invention relates to an improvement in a method for producing IFN-α by culturing IFN-α producing cells in a medium, and aims to enhance the production of IFN-α.

[Prior art]

IFN-α is generally produced by cells such as white blood cells induced by viruses, and is said to be useful against various diseases caused by viruses.

However, in order to use IFN-α for the prevention or treatment of viral diseases, it is necessary to collect a large amount of IFN-α.

Conventionally, methods of adding carboxylic acid, polyhydric alcohol, or glucocorticoid to a culture medium have been proposed as methods for mass-producing IFN-α.

However, the reality is that even with this method, a satisfactory production amount cannot be obtained.

[Problem that the invention seeks to solve]

Therefore, an object of the present invention is to enhance the production of IFN-α.

[Structure of the invention]

From this point of view, the present inventors have conducted various studies and found that the amount of IFN-α produced can be enhanced by bringing IFN-α producing cells into contact with a lower alcohol before induction.

The present invention was completed based on such new findings, and the gist thereof is a method for producing IFN-α by culturing IFN-α producing cells in a medium, in which the I
The present invention relates to a method for producing IFNTα, which comprises contacting FN-α producing cells with a lower alcohol.

The IFN-α-producing cells used in the present invention are IFN-α-producing cells.
There is no particular restriction as long as it can produce α, such as cultured lymphoblastoid cells. Preferred IF
Examples of N-α producing cells include cultured lymphoblastoid cells from the viewpoint of production ability. As the cultured lymphoblastoid cells, Namalva strain is particularly preferred. Namalva stock is 20
This cell line is selected from the lymphocyte lines of Burkitt's lymphoma and leukemia patients, and it is currently believed that this cell line best produces IFN-α (Inte
r, J. Cancer, IL 327 (197
3), J.

C11nical Microbiol, 1+ 1
(1975)).

Contact between IFN-α producing cells and lower alcohol causes IF
The contact is carried out before the induction of N-α, and the contact is usually carried out by culturing in a medium supplemented with lower alcohols, and the lower alcohols are removed during IFN-α induction, but the lower alcohols present at the time of induction are There is no problem. In addition, when replacing the medium, use a lower alcohol-containing solution (as a solvent, in addition to the medium described later, PBS (-) and Hank's solution are exemplified). ] can also be carried out by temporary contact with

When contact is carried out by adding lower alcohol to the medium,
RPM11640 medium is commonly used as the medium, but Eagle's MEM medium, DEM medium, H
am's F-12 medium, RITC56-2 medium, etc. can be used, preferably neonatal bovine serum at 0.1 to
15 (V/v)%, more preferably 10 (V/V)%
Eagle's RPM11640 medium supplemented with
MEM medium, DEM medium, llam's F-
12 medium, human albumin 0.1-1% (W/V)
An example is RITC56-2 medium to which a certain amount has been added.

The culture conditions during contact with lower alcohols are, for example, as follows: Usually, the cell concentration is about 5-6 x 105 Ce11/ml, the culture temperature is 30-40°C, preferably about 37°C, and the culture time is 1-4. 1 day, preferably about 2 to 3 days. At this time, the final concentration of lower alcohol should be sufficient to enhance the production of IFN-α (usually 0.1 to 3
V/V)%, preferably 0.1 to 1 (V/V)%
That's about it. Generally, the amount of IFN-α produced increases as the concentration of lower alcohol increases, but when it exceeds 3 (V/V)%, the amount of IFN-α produced decreases due to the toxicity of alcohol.

Lower alcohols include methanol, ethanol, n-
Examples include propatool, but ethanol is particularly preferred.

Furthermore, it is preferable to add glucocorticoids (eg, dexamethadone, hydrocortisone, etc.) in addition to alcohol. At that time, lower alcohol [final concentration 0.1
~3 (V/V)%] and simultaneously add glucocorticoid to a final concentration of about 10-7 to 10-4M.

IF subjected to contact treatment with lower alcohol according to the present invention
N-α-producing cells may be induced by a conventional method (for example, induction with a virus such as Sendai virus), and the desired IFN-α produced from the cells may be separated and collected.

As for the culture conditions at that time, the same medium as above may be used, and the cell concentration may be, for example, 2
It is preferably about 106 cells/ml, and the inducing agent is preferably about 100 HAU/ml, for example, in the case of Sendai virus. The culture time is about 20 to 24 hours, and the culture temperature is about 25 to 37°C.

The concept of an example of the IFN-α production method according to the present invention is as follows: The cell concentration is 5 to 6×105 cells/ml, and 10(V/ml).
V) RPM1164 supplemented with neonatal bovine serum at a rate of %
Culture is started at approximately 37°C using 0 medium.

On the 2nd to 3rd day of culture, add 0.1 to 3 (v) lower alcohol.
/v)% and further heated to 48~7 at around 37℃.
Incubate for 2 hours.

After that, the medium was replaced with fresh RPM11640 medium, adjusted to 20x10s cells/ml, and Sendai virus was added to a final concentration of about 100 HAU/ml.
IFN-α was induced for 20-24 hours at 7°C, and the supernatant was subjected to IF.
N-α titers are determined by the FL cell-5indbis virus combination cytopathic inhibition method.

〔effect〕

According to the present invention, IFN-α producing cells are cultured to produce IFN-α.
In producing -α, the productivity of IFN-α is significantly increased, and it is useful for industrial production of IFN-α.

Example 1 Effect of Ethyl Alcohol Addition Strain GC8201, which was isolated and established from Namalva cells as a strain that produces IFN-α at a particularly high rate, was used in RPM11640 medium supplemented with 10 (V/V)% neonatal bovine serum. Culture in 250ml spinner flasks. On the third day after the start of culture, the cell concentration was approximately 150 x 104 cells/
ml, add ethyl alcohol to a final concentration of 0.
It is added to each spinner flask at a concentration of 1 to 3 (V/V)% and further cultured for 48 hours. After this, cells were separated by centrifugation (1500 rpm, 5 minutes), RPM
Using 11640 medium, suspend the cell pellet at a cell concentration of 20.
Adjust to 0x104 cells/ml. Next L
Final injection of Sendai virus to induce FN-α
Add so that the amount becomes 0.00 HA/ml. This is 37
Incubate at ℃ for 20 hours and centrifuge (3000 rpm).
, 5 minutes), the IFN-α titer contained in the supernatant is measured. The results obtained are shown in Table 1.

Table 1 Example 2 IFN-α is produced according to the method of Example 1.

However, after first determining the optimal concentration of dexamethasone, the effect of combined use with ethyl alcohol was investigated. The results are shown in Table 2.

(Left below) Table 2 Since the addition effect was observed at 10-8M or higher, the dexamethasone concentration was set to 10-7M and the ethyl alcohol concentration was varied. The results are shown in Table 3.

(Margin below) Table 3 Example 3 After determining the optimum concentration of hydrocortiscin following the method of Example 2, the effect of combined use with ethyl alcohol was investigated.

The results are shown in Table 4.

Table 4 Similar to dexamethasone, the addition effect was observed at 101M or more, so the hydrocortiscin concentration was set to 10-7M and the ethyl alcohol concentration was varied. The results are shown in Table 5.

(Margin below) Table 5 Procedural amendment (own ship) September 25, 1980

Claims (4)

[Claims] (1) A method for producing interferon-α by culturing interferon-α-producing cells in a medium, which comprises contacting the interferon-α-producing cells with a lower alcohol before induction.
Production method of α. (2) The method for producing interferon-α according to claim (1), wherein the lower alcohol is ethanol. (3) Contact with a final concentration of lower alcohol of 0.1 to 3 (
% v/v)% medium for producing interferon-α according to claim (1) or (2). (4) Claim (1) characterized in that a glucocorticoid is used in addition to a lower alcohol.
, (2) or (3).