JPS59179095A - Production of interferon - Google Patents

Abstract

PURPOSE:When interferon is produced by stimulating plate-cultured human cells with a complex of polylipoinosic acid and polylipocytidylic acid, the ionsinic residue in the complex is kept more than a specific % based on the total uncleoside residues to permit the high-yield and stabilized production. CONSTITUTION:Interferon is produced by stimulating plate-cultured human cells, preferably human cells from foreskins, with a complex of polylipoinosinic acid and polylipocytidylic acid. At this time, the inosinic residue in the complex is kept more than 55mol% based on the total nucleoside residues, thus resulting in stabilized and high-yield production of interferon.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel method for producing interferon.

Interferon is expected to be used as a medicine due to its antiviral and antitumor activities, and is attracting attention.

Intertubolon is species-specific, and interferon can only be effectively produced in humans by human-derived cells.

With the recent development of genetic technology, the technology to integrate cloned genes into Escherichia coli and produce large amounts of interferon is now being established, and industrial production of human/J-interferon is being established. has not yet been completed.

In the conventional method for producing human β-interferon using human diploid cells, a polyriboinononic acid/polyriboinotic acid complex (hereinafter referred to as "poly(I) poly(CI)") is used as an interferon inducer. The use of poly(1)poly(C) with high inducing ability has great practical value.

In such a method for producing interferon using poly(I) poly(C), when producing interferon using the microcarrier method, poly(I) poly(C)
) is the mole fraction of hypoxanthine base in poly(I) acid (hereinafter referred to as "poly(I)J") or the mole fraction of hypoxanthine base in polyphtidylic acid (hereinafter referred to as "boI CI"). A method of increasing interferon production by using a product that exceeds the above range (Special 15+L 57-
2.220) is known. On the other hand, plane +□y
In producing interferon using the i-nutrient method,
It was believed that a molar ratio (I/C ratio) of hypoxanthin base in polyfil to nodonone base in poly(C) of 1 indicates the highest interferon production (M'l R
e-patent publication).

As a result of various studies for the purpose of preparing poly(1) poly(C) with a high ability to induce interferon production in a planar culture method, the present inventor has discovered that, contrary to conventional knowledge, interferon production can be induced in a planar culture method. /In the production method, poly(
I) When poly(C) is used which has a mole % or more of 55% or more of its inosine residues aligned with all 7 creondo residues, a relatively low concentration of BoIJ (I) Pol) (C) is used. At practical concentrations, as in the case of A IIA degree, 1? ) The present invention was completed based on the finding that high interferon production can be stably obtained.

That is, the method of the present invention is a method for producing interferon by puncturing human cells cultured on a flat surface with poly(I) poly(C), in which all 7 creon residues of the inonone residues are used as poly(J) poly(C). This is a method for producing interferon characterized by using a compound whose mole % based on the residue is 55% or more.

A method for producing interferon using a planar culture method is known, and in the method of the present invention, poly(1) bol) (C) having an I/C ratio of 1'ν constant is used as an interferon inducer.
There are no particular restrictions on other method conditions other than the use of. Regarding the interferon/production method using such a planar culture method, for example, see "Progress in Research on Proteins, Nucleic Acids, Enzymes - Interferon," published by Kyoritsu Shuppan Co., Ltd., January 1982, pp. 49-60. clarification of the description. The flat culture method is based on the adhesion dependence of human cells (
This refers to a method of culturing the cells without adhering them to the surface of a substrate, and as long as the method is based on the principle of culture, any type of culturing apparatus may be used.

That is, interferon-producing cells include human fetal lung-derived cells (HEL299.HA AIN55), human foreskin-derived cells (FS4, Flow, 7000),
Human tumor-derived fibroblasts (MG63) are applied. It is particularly preferable to use cells derived from human foreskin.

Also, the selection of cell culture media, interferon production media, priming, super-induction treatments, and. - Setting of each method condition can be determined as appropriate based on preliminary experimental parsimony i and I, respectively. Various other technical modifications that can be adopted in the 12-sided culture method can be applied as long as they do not conflict with the purpose of the method of the present invention.

The greatest feature of the present invention is that poly+1)po')<C+ is used in an amount of 55% or more by mole of all 7 creond residues of inonone residues.

Such poly(I,) and poly(C) can be prepared by any purposeful method, including poly(I)(Ii and poly(C)) of appropriate molecular weights at appropriate concentrations and in desired molar ratios. or poly(J,'+poly(C) with I/C of 1).
) may be suitably added to poly(I). The concentration of BoIJ (1) (, C) used is usually 5-
It is 100 μM.

It goes without saying that in the method of the present invention, there are no particular restrictions on the method of observing the produced interferon, and this step can be carried out by applying the knowledge of ordinary interferon purification techniques of those skilled in the art. In order to clarify the structure and effects of the method of the present invention, an example of the method of the present invention is shown as an example.

Example l Plastic tissue culture with a diameter of 60n/Jarenich l
- Diploid cells (Flow 7000) 5 x 10
The cells were added to 5 ml of E-Cl M EX mouthwash containing 10% of 10% of blood/1li and cultured at 37°C for 1 week.

The culture solution was removed, and 100 IU of interferon (1 cut edge t11 position) /'rtt (and 5 ml h4 EM medium containing 2.5% baby serum) It // Yare was added.
j, After continuing the culture at 37°C for about 20 hours, the culture medium was removed again.

Each 17C ratio (I) -flJ (C) is 2
5 μM again i;! Add 100 μM #degree and incubate at 37°C for 1
Cultured for hours. The culture solution containing poly(1) poly(C) was removed, Mueagle MEM medium containing 25 μF II+/cyclohequinomide was added, and cultured for 3 hours and 30 minutes. The cells were treated for 1 hour and 30 minutes.
After washing the cells with a saline solution (Earle's solution), 2 ml/Petri dish of Eagle's MEM medium containing 2.5% fetal Uno serum was added and cultured at 37°C for 24 hours. The final produced interferon in the culture medium is transferred to FL cells and V
esicular 5toynatilis viru
The micro-dye uptake method using s was determined and converted to international w position. The results are shown in Table 1.

In addition, in the table, poly(I)% means poly(1) poly(
C) is the mole percent of ynone residues to total nucleosotho residues in C). The interferon production amount is the geometric mean value of experimental samples obtained using 5 Petri dishes.

U211111111 Table 1 *Poly(1) Poly(C) Usage Concentration Example 2 Same as Example 1 using FS4 cells instead of FIO~V7000 as interferon producing cells. produced interferon. The results are shown in Table 2.

Table 2 * Poly(1) Usage concentration example of poly(C) 3 Poly(■) as poly(CI)/poly(C): Poly(C)
I/RI feron was produced in the same manner as in Example 1 using a molar ratio of 1:1.2°1 or 9.1. The results are shown in Table 3.

Table 3 *Interferon-producing cells

Claims (1)

[Claims] In a method for producing interferon by stimulating flat-cultured human cells with a polyriboinononic acid/polyribocytinyl acid complex, the molar percentage of the ynonyl residues relative to the total nucleondo residues as the polyriboinononic acid/polyribontidylic acid complex is 5596 or more. 1. A method for producing interferon, characterized by using a certain one.