JPS6018399B2 - Efficient production method of interferon - Google Patents

Description

[Detailed Description of the Invention] Interferon (hereinafter abbreviated as IFN) is produced by culturing leukocytes, lymphoblastoid cells, seniblastocytes, human membrane cells, etc., and after reaching a certain cell concentration, the cells are Isolated or not isolated, induced with various inducers such as viruses or synthetic double-stranded RNA (poly l:C),
Methods of inducing FN are known.

Antimicroboa IAge is secreted from cells several hours after treatment with an antimicrobial agent, and its titer is thought to reach a certain value after a certain period of time (Antimicroboa IAge).
nt and Chemo eraphy, 20(1)
5, 1981).

Based on these findings, conventionally, except for N production by spontaneously producing cells, N was collected only once every time it was secreted into the medium. As a result of various studies on the growth physiology of the cells used and the culture conditions, the present inventors found that
When we isolated cells that seemed to have lost their IFN production capacity from the culture system, added them to fresh beach soil to which appropriate nutrients had been added, and cultured them in suspension, surprisingly, the cells disappeared again. The application was filed on the same day as the present application, demonstrating the ecological power of N production and discovering the fact that it produces a significant amount of N.

As a result of further studies on the growth physiology and culture conditions of the cells used, the present inventors discovered that, without necessarily isolating and recovering the cells, they removed the culture medium from the culture medium containing the cells, and instead added fresh cells. They found that by adding diluted salt, the N-producing ability of the cells was maintained without loss, and that a large amount of N was produced, and the present invention was completed.

To explain the method for obtaining mN according to the present invention, for example, when lymphoblastoid cells are used, first a high concentration culture medium is prepared for the cells.

At this time, the higher the cell concentration, the more efficient it is up to a certain concentration. Various efforts have been made to obtain a highly concentrated cell culture solution. In the present invention, the highly concentrated liquid is prepared by a new method, and the method is shown in the reference example, but the details are described in a new application filed by the same applicant on the same day as the present application. It is detailed in ``High Concentration Cultivation Method and Its Secret Demon''. . Next, the obtained cell fluid is diluted or the cells are isolated, suspended in a fresh medium at a concentration suitable for N production, and treated with an inducing agent. It is more effective to treat the cells with a treatment agent prior to treatment with the inducing agent, collect the cells singly, and resuspend them in fresh soil. Immediately after the start of culture, or after a certain period of time, the culture medium is collected from one side and fresh medium is added from the other side to continue the culture, so that the cells continue to produce N for a long time, and depending on the number of cells per cell used. Compared to the one-batch method, it became possible to produce a chopstick's worth of IFN. As the medium for N production used in the present invention, RPMI-1640 Wakachi medium supplemented with penicillin, streptomycin, glutamine, Hebes buffer, etc., and Eagle's MEM medium are suitable. 0.5-20%, preferably 3-10%
% is suitable.

Furthermore, a serum-free medium is also effective in facilitating the purification and separation of interferon. As a serum-free medium, use insulin 0.3-10 instead of serum in the above medium.
0 mt', preferably 1 to 10 m/s, transferrin 0.1 to 100 mt/', preferably 1 to 1
0 Muta/[, selenite 0.1-100×10-7M
, preferably 0.5-10x10-7M, sodium pyruvate 0.1-100wM, preferably 0.5-20
Add mM and optionally serum albumin 0.1-200
A value of 1 to 50 m/s is suitable. Furthermore, various hormones, vitamins, etc. may be added. Regarding the treatment method using treatment agents, when lymphoblastoid cells are used, sodium petilate, dimethyl sulfoxide (hereinafter abbreviated as DMSO), vitamin A,
More preferable results can be obtained by treating with a treatment agent such as 5-promodeoxyuridine alone or in combination.

After the cells were treated with the above-mentioned treatment agent, they were collected aseptically and resuspended in a fresh medium.
0 HAu/). Cell concentration is 1×2
It may be within the range of 0x16 cells, but preferably 4 to 7x1 cells/similar. In Senblast cells, synthetic double-stranded RNA (poly l:C)
In some cases, this is followed by hyperstimulatory treatment (for example, a combination of cyclohexamide and actinomycin ○).

After these transfer treatments, the cells are cultured for 18 to 4 hours under 2 to 370℃ to produce interferon.

During continuous culture, it is convenient to use a device such as the one shown in the reference example to drain only the medium supernatant from one side and add fresh medium from the other side. These devices and operating methods are filed on the same date and by the same applicant. It is detailed in the new application ``Title of the Invention: High-concentration culture method of suspension culture and its packaging.'' The outline of the process is as follows: A high-concentration culture device for suspended cells is used, which has a gate at the bottom of the culture tank, a culture outlet tube that also serves as a cell sedimentation tube, and a conduit for adding fresh medium. Culture may be carried out while adding the medium from the medium through the medium conduit and draining the culture supernatant from the medium supernatant discharge tube.

The apparatus used is shown in FIGS. 1 to 3.

Example 1 Using the high concentration suspension culture device described in the reference example, 3%
Namalva cells were cultured in RPMI-164 Seitakuchi containing calf serum to obtain 6 x 1 cells/500 cells.The cells were centrifuged at 30 pm for 10 minutes in a sterile body. was suspended in serum-free beach 500' as shown in Example 1, lmM/dium butyrate, 1% v/v DMS.
The cells were then cultured at 37°C for 22 hours using the same apparatus.

After culturing, the cells were collected by centrifugation again and placed in the same serum-free medium 5.
00' floating. Transfection was carried out with Sendai virus so that Au/secretion was obtained for 500 days, and after culturing at 370°C for 8 hours, the cells were transferred at 28:00.

From this point, add fresh serum-free medium for 15-20 [/h]
The cells and the medium supernatant were separated at the same speed using a cell sedimentation tube, and then taken out of the system. Interferon was recovered in this supernatant. Interferon titers are shown below every 2 hours. In addition, the mN production amount of 6 × 1 cells/' in one batch was 42,000
It was u/w'.

Therefore, the optimal conditions last longer than in the case of one batch, and because the production was carried out for a long time, the I
I was able to get FN. Reference Example A cylindrical tube having a volume of 100 tubes with a diameter of 2.5 tubes was used as a precipitation tube in a culture tank of 1 tube (diameter: about 132 tubes, height: about 24 tubes) as shown in FIG.

RPMI-1640 (added with 3% calf serum) was used in the culture tank, and Namalva cells, which are lymphoblastoid cells, were inoculated at 7×1 cells/cell as seeds. Supernatant discharge and fresh column addition were carried out at a rate of 10.5 secretions/hr for a total volume of 500 cm. The pump used was a veristake pump. Culture was carried out at 370°C with a fly frame rotation speed of 5pm and aeration of 16°/min. The results of measuring the number of cells using a hemocytometer are shown in FIG. In addition, 12
After q hours, the cell concentration reached 6.6×1 cells/secretion, and the survival rate was 96.5%. If the cell sedimentation tube used for comparison is not used, 2.2×
Cell growth stopped after 1 pic.

[Brief explanation of drawings]

FIG. 1 shows a modified spinner flask (main body made of glass). 1 is a glass cell sedimentation tube (exhaust port), 2 is a glass column addition port, 3 is a stainless steel shaft, 4 is a Teflon sampling tube, 5 is a stainless steel air inlet,
6 is a stainless steel air outlet, 7 is a Teflon rotating shaft, 8 is a Teflon rotor, 9 is a silicone stopper,
10 represents a cap made of heat-resistant plastic, and 11 represents a hammer opening. The capacity of 1 is about 120 [, the caliber of 2 and 3 is about 7 winds, 4,
5 and 6 have a diameter of about 3 feet, 7 has a diameter of about 19 capes and a height of about 3 feet, 8 has a diameter of 12.5 and a length of about 75 legs, 1 and 1 have a diameter of about 4
2 willows, about 37.5 skins long. FIG. 2 is a plan view of the modified spinner flask. FIG. 3 shows an example of a large-scale culture tank and a high-density cell culture device including the same. 12 shows the culture tank main body, 13 shows the cell sedimentation tube (discharge pipe), 14 shows the neck azusa wing, 15
1.7 is a pump for discharging medium, 16 is a pump for adding medium.
18 shows an addition tower base tank and a sterile filter. FIG. 4 shows the relationship between the culture time (axis) and the number of viable cells (vertical axis) in Example 1. Solid line 1 represents the number of live cells obtained by the method of the present invention using a cell sedimentation tube, and dotted line 2 represents the number of live cells obtained by the conventional culture method. Figure 1 Figure 2 Figure 3 Figure 4

Claims (1)

[Claims] 1. Animal cells induced with an interferon inducer are cultured by continuously adding fresh medium to the culture medium and separating the culture supernatant, and interferon produced from the separated supernatant is collected. An efficient method for producing interferon.