JPS645879B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing interferon, and more particularly to a method for producing interferon by treating animal cells grown on microcarriers with an interferon-inducing agent. Conventionally, methods for culturing tether-dependent cells such as diploid cells using Lou bottles or roller bottles are known, but it is thought that it is quite difficult to culture large amounts of tether-dependent cells using this method. It is being In other words, in this method, cells only grow in a single layer on the bottom of the roux bottle or the inner surface of the roller bottle, so it is necessary to handle a very large number of roux bottles or roller bottles for mass culture. This is because it becomes extremely complicated and it is considered almost impossible to control culture conditions such as pH and dissolved oxygen concentration in the medium at a constant level. Recently, a culture method called microcarrier culture method suitable for mass culture of tether-dependent cells has been developed.
In this culture method, seed cells are inoculated into a medium containing suspended microcarriers with positively charged chemical residues, collagen-coated microcarriers, or other microcarriers capable of adhering cells. This is a method of culturing in the same condition. The inoculated cells attach to the microcarrier surface and proliferate there. The microcarrier culture method can be handled in large quantities, and the culture conditions can be easily controlled at optimal values, so it can be said to be a culture method suitable for scaling up to industrial production scale. On the other hand, a technique for producing interferon in cultured cells is called the superinduction method, in which cells are stimulated with an interferon inducer and then treated with a metabolic inhibitor such as cycloheximide or actinomycin D. Methods for further enhancing interferon production are known. The present inventors have focused on the above-mentioned microcarrier culture method and superinduction method as methods for producing large amounts of interferon derived from cultured cells such as diploid cells, and have also focused on the PH of the culture and interferon production process. Attempts have been made to produce interferon by controlling the temperature to an optimal value and aerating air or oxygen gas into the gas phase or liquid phase to an extent that the dissolved oxygen concentration in the medium does not become zero. However, with this method, it is not possible to stably produce a high unit of interferon, and often only a low unit of interferon is produced. An object of the present invention is to solve this problem and provide a method for producing interferon which is highly effective in stably producing a high unit of interferon. The purpose of the present invention is to reduce the dissolved oxygen concentration to 10% or more relative to the saturated solubility in air during interferon production in the microcarrier culture method.
By controlling the dissolved oxygen concentration at a constant level of 50%, preferably in the range of 15% to 35%, it is preferable to maintain the dissolved oxygen concentration within the range of 10% to 50% of the saturated solubility in air even during cell proliferation by microcarriers. This is achieved through constant control. Next, a method for culturing tether-dependent cells such as two-culture cells and producing interferon using a microcarrier culture method, which is a premise of the present invention, will be explained. Microcarriers are suspended in a medium, seed cells are inoculated into the medium, and culture is carried out by controlling the medium to an appropriate pH temperature while gently stirring. As microcarriers, polysaccharides such as
An appropriate amount of amine residues are bonded to microparticles made of dextran, dextrin, starch, cellulose, their substituent derivatives, etc., or certain synthetic polymers, such as polyvinyl alcohol, hydroxy-substituted acrylates or methacrylates, polystyrene, etc. Those that have been made are used. The microcarrier concentration is determined by the microcarrier material,
1 ml of medium, depending on the type of cells to be cultured
The microcarrier surface area per area is approximately 10 cm ² ~
A concentration range of 100 cm ² is preferred. Culture composition, serum concentration, etc. are appropriately selected depending on the type of cells to be cultured, cell concentration, etc. During the culture, the medium is replaced as appropriate, and the culture is continued for several to 10 days until the surface of the microcarriers is almost completely covered with cells. Before the proliferated cells are treated with an interferon-inducing agent, it is preferable to add a low unit of interferon and a negatively charged water-soluble polymer and to incubate the cells for several hours to one day. Techniques for treating cultured cells with an interferon inducer to produce interferon include a method of stimulating cells to produce interferon using an interferon inducer such as natural or synthetic RNA, and an interferon method called the calcium method. A method using an interferon inducer such as double-stranded RNA called the super induction method, in which an appropriate amount of calcium is added to the culture medium at the time of using an eron inducer, or 10 hours before, to further enhance interferon production. After stimulating the cells, interferon production is further enhanced by treating the cells with a metabolic inhibitor such as cycloheximide or actinomycin D (US Pat. No. 3,773,924), using an interferon inducer called the UV method. Methods are known, such as a method in which cells are irradiated with ultraviolet rays for several hours before and after cell stimulation to further enhance interferon production. The present invention preferably maintains the dissolved oxygen concentration in the medium during the above-mentioned interferon induction treatment, including during cell culture, in the range of 10% to 50%, preferably 15% to 35% of the saturated solubility. is the essence. The method of the present invention is particularly effective for induction treatment using the superinduction method. It is preferable that the dissolved oxygen concentration in the present invention is always controlled to be constant within a specified concentration range. If it is necessary to replace part of the medium during culture, it is desirable to adjust the dissolved oxygen concentration of the replacement medium to the dissolved oxygen concentration during culture in advance, but it may be adjusted with nitrogen, etc. immediately after replacement. do not have. As the culture solution used in the present invention, any conventional culture solution can be used, but Eagle's MEM medium, Dulbecco's MEM medium, etc. are particularly preferably used. The present invention will be specifically explained below with reference to Examples. Examples 1 to 7 and Comparative Examples 1 to 3 Newborn calf serum 5%, crosslinked dextran microcarriers having diethylaminoethyl groups 3 g/
Eagle MEM1 containing human normal diploid cells were inoculated at a rate of approximately 1.2 × ¹⁰ cells/ml in 11 glass spinner flasks at 37°C with gentle agitation.
Cultured at pH 7.2 for 6 days. Regarding the 10 flasks, the dissolved oxygen concentration during cultivation was controlled at an appropriate value in the range of 7% to 75% of the saturated solubility, and the other flask was cultured with aeration so that the dissolved oxygen concentration did not reach zero. Medium was exchanged on the 1st, 3rd, and 5th days, and nitrogen was aerated after each medium exchange to quickly reduce the dissolved oxygen concentration to the desired concentration. The number of cells reached was around 1×10 ⁶ cells/ml in all cases. Next, interferon 100 international units/ml,
Eagle, containing carboxymethylcellulose
The medium was replaced with MEM medium and incubated at 37°C, pH 7.2 for about 20 hours. During this period, the dissolved oxygen concentration was controlled at the concentration during each culture except for one. Then 10 μg/ml cycloheximide and 50 μg/ml
g/ml poly():poly(C) was added and incubated at 37°C for 4 hours, and further 4 μg/ml actinomycin D was added and incubated at 37°C for 1 hour. Thereafter, the medium was exchanged twice with Eagle's MEM medium supplemented with 0.5 g/methylcellulose, and incubated at 37°C for about 2 days. The dissolved oxygen concentration during that time was also controlled at the same value as the concentration in the medium, except for one. The amount of interferon produced in the final medium used was measured using FL cells and V. stomateis virus.
It was measured using the CPE inhibition method and converted to international units. The results are shown in Table 1 and Figure 1.

[Table] Figure 1 shows the pH of Eagle MEM medium 1 containing microcarrier concentration 3g/V% and calf serum 5V/V%.
7.2 The results are shown in which human diploid cells were cultured to produce interferon under controlled conditions such that the temperature remained constant at 37°C. The dissolved oxygen concentration in the medium was controlled to be constant at various concentrations. As is clear from Figure 1, when cultured in a medium in which the dissolved oxygen concentration in the medium is controlled to be less than 10% or more than 50% and treated to induce interferon, only a small amount of interferon can be produced. It is possible to obtain high units of interferon production if the cells are cultured in a medium controlled at a constant value in the range of 10% to 50%, preferably 15% to 35%, and treated to induce interferon. be. Examples 8 to 10 and Comparative Examples 5 to 6 Newborn calf serum 5V/V%, crosslinked dextran microcarriers having diethylaminoethyl groups 3
Human normal diploid cells were inoculated at a rate of approximately 1.2 x 10 ⁵ cells/ml into Eagle MEM1 containing 1.5 g/ml, and incubated at 37°C PH with gentle stirring in a glass spinner flask.
7.2 for 6 days. The culture was carried out with aeration so that the dissolved oxygen concentration during the culture did not become zero. then 1
The culture solution was dispensed into 5 small glass spinner flasks in 200 ml portions, replaced with Eagle's MEM medium containing 100 international units of interferon/ml carboxymethyl cellulose, and incubated at 37° C., pH 7.2 for about 20 hours. During this time, the dissolved oxygen concentration in the medium was controlled at 20%, 30%, 50%, 75%, and 80%, respectively. Next, 10 μg/ml cycloheximide and 50 μg/ml Poly():poly(C) are added and incubated under the same conditions for 4 hours. Further 4μg/
Add ml of actinomycin D and incubate for 1 hour in the same manner. After that, the medium was exchanged twice with Eagle's MEM medium containing 0.5 g/methylcellulose, and in the same manner at 37°C, PH7.2, dissolved oxygen concentration 20, 30,
Incubate for 48 hours at 50, 75, and 80%. The amount of interferon finally produced in the medium used was measured by the CPEinhibition method using FL cells and Vescuel stomateisis virus, and converted to international units. The results are shown in Table 2 and Figure 2.

[Table] Figure 2 shows the pH of Eagle MEM medium 1 containing microcarrier concentration 3g/V% and calf serum 5V/V%.
7.2 Culture human diploid cells by controlling the temperature to be constant at 37℃ and aerating so that the dissolved oxygen concentration does not reach zero.
This shows the results of interferon induction treatment by dispensing it into a book, controlling the dissolved oxygen concentration at various values under the same PH temperature conditions. From Figure 2, dissolved oxygen concentration is 10% to 50%, preferably 15% to 50%.
It can be seen that it is possible to obtain a high titer of interferon production when interferon is induced by controlling it at a constant level within the range of 35%.

[Brief explanation of drawings]

FIG. 1 shows the results of Examples 1 to 7 and Comparative Examples 1 to 3. The vertical axis shows the interferon titer (international units/ml), and the horizontal axis shows the dissolved oxygen concentration in the medium. Figure 2 shows Examples 8 to 10 and Comparative Example 5.
-6 results are shown. The vertical axis shows the interferon titer (international units/ml), and the horizontal axis shows the dissolved oxygen concentration in the medium during interferon induction treatment after dispensing.

Claims (1)

[Claims] 1. When treating animal cells grown on microcarriers with an interferon-inducing agent to produce interferon extracellularly, the dissolved oxygen concentration in the culture medium should be adjusted to 10% or more of the saturated solubility in air.
A method for producing interferon characterized by keeping it within 50%.