JPH0724595B2 - Method for producing cytokine - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing a cytokine. More specifically, the present invention relates to a method of producing a cytokine, characterized in that the induction step and the production step are carried out under different temperature and pH values.

(Prior Art) Cytokines play an important role in the interrelation between regulation of cell function and tissues of the body.

The term “cytokine” refers to any molecule or group of molecules that has a biological activity produced by living cells and secreted around them. Typical examples thereof are intoleukin, interferon (IFN-S), lipocortin, and tumor necrosis factor.

Living cells can also produce cytokines in vitro by the action of appropriate inducers such as viruses, lectins, mitogens, dexamethasone, phorbol esters, ionophores, and synthetic ribonucleic acids. Therefore, the biologically active substance can be produced on an industrial scale by using an appropriate method.

Cytokines are also made using so-called natural methods, ie genetic engineering. In this genetic manipulation, when an inducer is added to human or animal cells, the cells are acted upon by the inducer to produce cytokines in the nutrient solution. This cytokine is removed from the medium.

There are the following reports regarding this method.

For example, lipocortin using glucocorticoid [B. Rothhut et al .: FEBS Lett. 219 , 169 (1987)], PMA
B cell stimulating factor (IL-4) by use [J.Hu-Li et al .: Journal of Experimental Medicine (J.Exp.Med.) 165 , 157 (1987) )], Interleukin-2 [K.Kato et al., Using concanavalin A: Biochemical Biophysical Research Communication (Biochem.Biophys.Res.Commun.) 127 , p. 182 (1
985)], α-interferon using Sendai virus [K. Cantell et al .: Methods Enzymol. 72 , 29
Page (1981)] and so on.

A feature common to each of the above methods is that cytokine induction and production are performed under the same conditions.

(Problems to be Solved by the Invention) An object of the present invention is to suppress the influence of factors that inhibit the synthesis of cytokines and increase the yield of cytokines.

(Means for Solving the Problem) The present invention recognizes that the product inhibitory action proceeds early after the optimal conditions for induction, and this can be avoided by appropriate restriction of ambient conditions. Is based.

The present invention, which was made based on the above recognition, is a method for producing a cytokine having a biological activity which is produced by an animal cell and is secreted around the animal cell. In a method comprising mainly two steps of an induction step and a production step for inducing cytokine production by adding one or more inducers, the temperature and pH value in the induction step and the temperature and pH value in the production step are different from each other. And a method for producing a cytokine characterized by:

By using the method according to the invention, the yield of cytokines from various cells in vitro can be considerably increased.

Cytokines are usually prepared by treating the corresponding cells with one or more inducers and then culturing them for a period of time. Then, the cytokine secreted in the nutrient solution is taken out by a usual method.

According to the present invention, the action of the inhibitory factor can be delayed and the yield can be considerably increased by changing the culture conditions at appropriate time intervals.

Observed, the biochemical effects that delay the synthesis of cytokines become more intense after the exponential interval of cytokine synthesis. Starting from an exponential time, cytokine production becomes less sensitive to changes in culture conditions, and
It is less disturbed than if the synthesis was delayed slowly.

The production interval can be extended by appropriate changes and selection of change points.

If no pH adjustment has been made, bring the pH value to a physiological value, ie 7.2-7.4.

When carrying out the method of the present invention, the culture temperature and culture pH value in the induction step are set to the culture temperature and
It is preferably higher than the pH value. In addition, it is preferable to adjust the culture pH 7 and the culture temperature in the production stage within the range of 15 to 40 ° C. Furthermore, after the start of cytokine production,
It is preferred to change the culture pH or temperature and to change the pH value or temperature during the exponential function of cytokine production.

(Example) Hereinafter, the method according to the present invention will be described in detail based on preferred examples. However, these examples do not limit the present invention.

Example 1 Improvement of human leukocyte α-IFN yield by changing pH value of culture medium Hemolysis was performed several times with 0.83% ammonium chloride solution,
Buffy coat (leukocyte-rich fraction obtained from fresh human blood) was purified from erythrocytes contaminated with impurities, and the resulting leukocytes were treated with mineral salts, glucose, and 1 mg / ml agammaglobulin-free human serum. Suspend at a density of 1 × 10 ⁷ cells / ml in the appropriate nutrient solution containing.

To the obtained solution, 200 IU / ml of human leukocyte α-IFN is added, and this mixture is incubated at 37 ° C. for 2 hours. next,
100 HA units / ml of Sendai virus is added to the cells and the mixture is incubated at 37 ° C. for 2 hours.

Tris-hydrochloric acid buffer is added to the bottle containing the cell suspension in an amount such that the pH value becomes 7 for 2 hours, and then the culture is performed for 20 hours. The nutrient solution is then centrifuged to remove the white blood cells.

The antiviral titer of raw IFN thus obtained was 2
It is 00,000 IU / ml. This value is twice as high as the titer of IFN obtained without adjusting the pH value with the buffer solution.

Example 2 Improvement of yield of human leukocyte gamma IFN and interleukin-2 by changing culture temperature and pH value Gamma IFN and interleukin-2 are simultaneously prepared in the same cell culture.

White blood cells obtained from fresh buffy coat or used during preparation of IFN are used for this production. A leukocyte suspension is prepared as described in Example 1.

White blood cells are suspended in a normal nutrient solution containing 1 mg / ml agammaglobulin-free human serum in a concentration range of 1 × 10 ⁷ to 2 × 10 ⁷ cells / ml.

10-20 μg / ml Concanavalin A, 1-10 μg / ml plant agglutinin, or 20-10 ng / ml mesereine
Or 0.5 to 2 μg / ml A23187 ionophore, or 5
Add ~ 30ng / ml phorbol myristate acetate and induce at 37 ° C.

Three hours after adding the inducer, the leukocyte-containing bottle is transferred to a constant temperature bath maintained at 30 ° C., the pH value is adjusted to 7, and then the mixture is cultured.

After culturing, gamma-IFN and interleukin-2 (IL
The cells are removed from the nutrient solution containing -2) by centrifugation.

The antiviral titer of gamma-IFN is 15,000 IU / ml and the antiviral titer of IL-2 is 1300 IU / ml. These values are 2-2.5 times higher than the titer of the product obtained at a constant temperature of 37 ° C without pH adjustment with buffer.

Gamma-IFN and IL-2 are separated by another purification operation.

Example 3 Enhancement of Lipocortin Yield by Changing Culture Temperature Buffy coats obtained from fresh human blood are purified from impure red blood cells by several hemolysis with 0.83% ammonium chloride solution.

Next, the obtained pure leukocyte fraction was suspended in a serum-free nutrient solution to a cell number of 1 × 10 ⁷ per ml, and further treated with 1 μM dexamethasone. Incubate at 37 ℃ for 2 hours. Then, the culture temperature is adjusted within the range of 30 to 32 ° C., and the culture is continued at that temperature for another 15 to 20 hours. It is then centrifuged to remove leukocytes from the nutrient solution.

The supernatant thus obtained, which is crudely prepared lipocortin, exerts a strong inhibitory effect on carrageenin-induced inflammation.

The anti-inflammatory effect of crude lipocortin produced without keeping the temperature constant is 2-2.5 times stronger than that of lipocortin produced at constant temperature.

Winter et al. Proc.Soc.Exp.Biol.Me Proceedings of Society for Experimental Biology and Medicine
d.), 111 , 644 (1962) and the carrageenin test method was used to measure the anti-inflammatory effect on rats.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (56) Reference JP 62-96083 (JP, A) JP 57-105195 (JP, A) Special table 57-501265 (JP, A)

Claims (5)

[Claims] 1. A method for producing a cytokine having a biological activity which is produced by an animal cell and is secreted around them, wherein one or more inducers are added to the animal cell corresponding to the desired cytokine. Then, in a method mainly comprising two steps of an induction step and a production step for inducing the production of cytokine, a cytokine characterized in that the temperature and pH value in the induction step and the temperature and pH value in the production step are different from each other how to. 2. A method for producing a cytokine, wherein the culture temperature and the culture pH value in the induction step are higher than the culture temperature and the culture pH value in the production step. 3. The method for producing a cytokine according to claim 1, wherein the culture pH and the culture temperature in the production stage are adjusted to 7 and 15 to 40 ° C., respectively. 4. The method for producing a cytokine according to any one of claims (1) to (3), wherein the culture pH or culture temperature is changed after the start of cytokine production. 5. The method for producing the cytokine according to claim 1, wherein the pH value or the temperature is changed while the exponential function of cytokine production lasts.