JPH0532031B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Field of Application] The present invention relates to interferon-α (hereinafter referred to as IFN-α).
-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 from cells such as white blood cells induced by a virus,
It 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 for mass-producing IFN-α have been proposed, such as adding carboxylic acids, polyhydric alcohols, or glucocorticoids to the culture medium. However, the reality is that even with this method, a satisfactory production amount cannot be obtained. [Problems to be Solved by the Invention] 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. I found out. The present invention was completed based on such new findings, and the gist thereof is to provide a method for producing IFN-α by culturing IFN-α producing cells in a medium, in which the IFN-α producing cells are cultured in a medium before induction. The present invention relates to a method for producing IFN-α, which comprises contacting IFN-α with a lower alcohol. The IFN-α producing cells acted upon in the present invention are:
There is no particular restriction as long as it can produce IFN-α, and examples thereof include cultured lymphoblastoid cells. Preferred IFN-α producing cells include cultured lymphoblastoid cells from the viewpoint of production ability. As the cultured lymphoblastoid cells, Namalva strain is particularly preferred. The Namalva strain is a cell line selected from 20 types of lymphocytes from patients with Burkitt's lymphoma and leukemia;
It is said to produce IFN-α most frequently [Inter.J.Cancer, 11, 327 (1973), J.Clinical
Microbiol., 1, 1 (1975)]. Contact between IFN-α-producing cells and lower alcohol is performed before IFN-α induction, and usually, the contact is performed by culturing in a medium supplemented with lower alcohol, and IFN-α induction Sometimes the lower alcohol is removed, but it can be present during induction. In addition, when replacing the medium, use a lower alcohol-containing solution (examples of the dissolved medium include PBS (-) and Hank's solution, in addition to the medium described below). ] can also be carried out by temporary contact with When contacting a medium with lower alcohol added, RPMI1640 medium is commonly used, but Eagle's MEM medium, DEM medium,
Ham'sF-12 medium, RITC56-2 medium, etc. can be used, and preferably newborn bovine serum is added at 0.1 to 15%
(v/v)%, more preferably RPMI1640 medium, Eagle's MEM medium added with about 10(v/v)%,
DEM medium, Ham'sF-12 medium, RITC56- with addition of human albumin at about 0.1-1% (w/v)
Examples include culture media. The culture conditions during contact with lower alcohols are, for example, as follows: Usually, the cell concentration is about 5 to 6 x 10 ⁵ cells/ml, the culture temperature is 30 to 40°C, preferably about 37°C, and the culture time is 1. ~4 days, preferably about 2 to 3 days. At this time, the final concentration of the lower alcohol should be an amount sufficient to enhance the production of IFN-α, and it is usually about 0.1 to 3 {v/v)%, preferably 0.1 to 1%.
(v/v)%. 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. Examples of the lower alcohol include methanol, ethanol, and n-propanol, with ethanol being particularly preferred. In addition to alcohol, glucocorticoids (e.g., dexamethasone, hydrocortisone, etc.)
It is preferable to add. At that time, lower alcohol [final concentration 0.1 to 3 (v/v)%] was added and glucocorticoid was added at a final concentration of 10 ^-7 to 3%.
It may be added to approximately 10 ^-4 M. The IFN-α producing cells subjected to the contact treatment with the lower alcohol according to the present invention are induced by a conventional method (e.g., induction with a virus such as Sendai virus), and the IFN-α producing cells are thus subjected to the desired production from the cells. What is necessary is to separate and collect IFN-α. As for the culture conditions at that time, the same medium as above may be used, the cell concentration is preferably about 2 x 10 ⁶ cells/ml, and the inducing agent is
For example, in the case of Sendai virus, it is 100HAU/ml.
degree is preferred. 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 method for producing IFN-α according to the present invention is as follows: The cell concentration is 5 to 6 × 10 ⁵ cells/ml, and 10 (v/ml).
v) Neonatal bovine serum was added to the proportion of %
Start culturing at approximately 37°C using RPMI1640 medium. Add 0.1 to 3 ml of lower alcohol on the 2nd to 3rd day of culture.
Add about (=/v)% and further increase the temperature to about 37℃ to 48~
Culture for 72 hours. After that, the medium was replaced with fresh RPMI1640 medium, adjusted to 20 × 10 ⁵ cells/ml, and Sendai virus was added to a final concentration of about 100 HAU/ml.
Induce IFN-α for 20-24 hours at 37°C, and remove IFN from the supernatant.
-α titer is measured by cytopathic inhibition method with FL cell-Sindbis virus combination. [Effect] According to the present invention, by culturing IFN-α producing cells,
When producing IFN-α, the productivity of IFN-α is significantly increased, and this method is useful for industrial production of IFN-α. Example 1 Effect of ethyl alcohol addition GC8201, a strain isolated and established from Namalva cells as a strain that produces IFN-α at a particularly high rate
The strain was added to RPMI1640 with 10 (v/v)% neonatal bovine serum.
Culture in a 250 ml spinner flask using medium. On the third day after the start of culture, when the cell concentration reached approximately 150 x ¹⁰⁴ cells/ml, ethyl alcohol was added to the final concentration.
It is added to each spinner flask at a concentration of 0.1 to 3 (v/v)% and further cultured for 48 hours. Thereafter, the cells are separated by centrifugation (1500 rpm, 5 minutes), and the cell pellet is suspended in RPMI1640 medium, and the cell concentration is adjusted to 200 x 10 ⁴ cells/ml.
Next, Sendai virus is added to a final concentration of 100 HAU/ml in order to induce IFN-α.
This was incubated at 37°C for 20 hours, and after centrifugation (3000 rpm, 5 minutes), the IFN-
Measure alpha titer. The results obtained are shown in Table 1.

[Table] 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.

【table】

[Table] Since the addition effect was observed at 10 ^-8 M or higher, the dexamethasone concentration was set to 10 ^-7 M and the ethyl alcohol concentration was varied. The results are shown in Table 3.

[Table] Example 3 After determining the final concentration of hydrocortisone following the method of Example 2, the effect of combined use with ethyl alcohol was investigated. The results are shown in Table 4.

[Table] Similar to dexamethasone, the addition effect was observed at 10 ^-8 M or higher, so the hydrocortisone concentration was increased to 10 ^-7 M.
and the ethyl alcohol concentration was varied. The results are shown in Table 5.

【table】

Claims (1)

[Scope of Claims] 1. A method for producing interferon-α by culturing interferon-α-producing cells in a medium, characterized by bringing the interferon-α-producing cells into contact with a lower alcohol before induction. A method for producing interferon-α. 2. The method for producing interferon-α according to claim 1, wherein the lower alcohol is ethanol. 3 Contact with the lower alcohol at a final concentration of 0.1 to 3.
(v/v)% in a medium
The method for producing interferon-α according to item 1 or 2. 4. Interferon-α according to claim 1, 2 or 3, characterized in that a glucocorticoid is further used in combination with a lower alcohol.
manufacturing method.