JPS60215632A - Preparation of erythropoietin - Google Patents

Abstract

PURPOSE:To obtain erythropoietin stably in a large amount at a low cost, by cultivating an established cell, having the ability to produce the erythropoietin, and derived from the human liver in a suitable culture medium to produce the titled substance in the culture. CONSTITUTION:An established cell, e.g. huH-2 strain, having the ability to produce erythropoietin (EPO), and derived from the human liver is cultivated in a suitable culture medium e.g. a synthetic culture medium containing 1-25% blood serum, preferably under conditions of low partial pressure of oxygen to produce EPO in the culture. The resultant culture fluid is collected and treated, e.g. salted out, dialyzed, filtered, centrifuged, concentrated, freeze-dried, etc. to give easily EPO (a substance which is a kind of hematogenic hormones, produced and secreted by the kidney, and acting on erythroblastic precursor cells to differentiate them into erythrocytes such as glycoprotein having 30,000-40,000 molecular weight). USE:A remedy for anemia caused by renal insufficiency, diagnostic agent or research reagent, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION Background of the Invention Technical Field The present invention relates to a novel method for producing erythroboetin (hereinafter referred to as EPO), and more specifically, to a method for producing EPO using a cell line derived from human liver cancer and having the ability to produce EPO. Concerning the law.

EPO is a hematopoietic hormone produced and secreted primarily by the kidneys, and is a substance that acts on erythroblast precursor cells to differentiate them into red blood cells, and is thought to be a glycoprotein with a molecular weight of 30,000 to 40,000. There is. EPO is useful for the treatment of anemia caused by renal failure (lack of EPO production ability), as a clinical diagnostic agent, and as a research reagent, and has a wide range of uses.

Prior Art GPO has traditionally been purified from the urine of patients with high EPO secretion, such as those with aplastic anemia. However, this method has a major drawback in that it is difficult to stably secure the necessary amount of urine as a raw material. Recently, as an alternative to this method, the following method for producing EP() has been proposed.

(1) A method of tissue culturing human renal cancer cells and collecting EPO from the culture (Japanese Patent Publication No. 42917/1983).

(2) A method of culturing human-derived phosphorus/e blastoid cells in the body of a warm-blooded animal and collecting EPO from the culture (Japanese Patent Application Laid-open No. 40411/1983).

(3) A method for collecting EPO from the urine of a healthy person (for example, the specification of Ryofu 1988-164.96).

However, as far as the present inventors know, these methods have not yet been put into practical use because they have various drawbacks (e.g., low yield, high cost). Therefore, there is a constant desire for a new method for producing EPO.

By the way, it is already known that certain liver cancer cells have the ability to produce EPO, and their primary culture
culture) EPO7 in the liquid! It has already been reported that l'pi will be born (T, 0kabe, et
al; TWelfthAnnual b'lee
ting-International 5ociet
y for )'1peri-mental l→e
matoloH: Annual eight lleeting
Abstractsl) 218 (1983))
is being done.

However, since these human hepatoma cells that have the ability to produce EPO are not established cell lines, subculturing in vitro becomes impossible in a short period of time, or EPO m
Since the vitality is lost, these cells are used for EP.
It has been impossible to produce O stably over a long period of time. On the other hand, no reports have been found regarding established cell lines derived from human liver cancer that have the ability to produce EPO.

SUMMARY OF THE INVENTION The present invention, in response to the above-mentioned desire, provides a method for producing Wr-standard EPO.

That is, the method for producing EPO according to the present invention uses established cell lines having EPO-producing order 1j derived from human liver cancer, such as huH.
This method is characterized by culturing the -2 strain, preferably under low oxygen partial pressure, and collecting EPO from the culture.

Effect The human liver cancer-derived EPO-producing cell line used in the present invention maintains a stable EPO-producing ability even after being cultured repeatedly over a long period of time.
O can be produced cheaply, in large quantities, and stably. According to one preferred embodiment (culture under hypoxic partial pressure - details below), K71 is present in the culture.
Since EPO is produced at 8 degrees, it is more advantageous to use El)O'
= Can be manufactured.

Although 41i1 cell culture technology has made unprecedented progress, it is still possible to easily establish individual cells while retaining their useful properties (EPO-producing ability in the context of the present invention). At present, this has not yet been achieved, so it was unexpected that EPO could be stably produced using human liver cancer-derived cell lines capable of producing EPO, as in the present invention. It should be said.

The established cell line used in the present invention has an EPO-producing cell line derived from human liver cancer. Specifically, the huH-2 strain established by the present inventors can be exemplified, but other human hepatoma cells capable of producing GPO may also be established and used according to the present invention. In addition, the apo-producing ability of human liver cancer-derived cell lines with EPO-sucking ability, including the huH-2 strain, was reduced by treatment with mutant extract JB (e.g., ethyl methanesulfonate, nitrosoguanidine, etc.), genes (e.g., EI from adenovirus)
It may also be possible to increase this by introducing A gene, etc.).

In the present invention, "established cell line-1" means an established cell line that can be stably subcultured in vitro over a long period of time.

huH-2 strain The hu H-2 strain, which was established by the present inventors as a cell line with EPO-producing ability derived from human hepatoma cells, has the following content. An attempt was made to deposit the huH-2 strain at the Institute of Microbial Technology, Agency of Industrial Science and Technology, but the deposit was refused.

(1) 0.5 cm6 of liver cancer cells surgically removed from a liver cancer patient with increased hematocrit value of huH-2 strain were shredded with scissors and washed with DM-160 medium (Kyokuto Pharmaceutical Industries). After that, it was mixed with DM16 containing 10% fetal bovine serum and antibiotics.
About 30 ml of 0 medium was made to be cloudy. Next, deisnose was added to this suspension at a concentration of 1000 U/ml, and the mixture was treated at 37°C for about 2 hours. After treatment, cells were collected by centrifugation and 0M containing the above serum and antibiotics.
After washing twice with 160 medium, the cells were suspended in this medium for 1 Mi. This suspension was placed in a 6 cm petri dish and cultured. Cultivation was carried out at 37°C with 5% CO2 introduced into the culture atmosphere. When culture was continued by replacing the medium with a new one every 3 days, the cells proliferated over the entire surface of the Petri dish 9 days after the start of culture.

After discarding the culture supernatant, the cells layered on the Petri dish were treated with trypsin (0.02%) to redisperse them, and the same procedure was repeated to repeat the culture, which resulted in unlimited subculture. A cell line capable of this was obtained. This cell line was named huH-2.

(2) Cytological properties aJ4fl cell morphology: epithelial-like (polygonal or irregular round)
b, Chromosome = diploid region (9: number of chromoplasts distributed from 44 to 80) C1 subculture: limited (subculture possible d, cryopreservation at 70°C and cumulative temperature in liquid chamber) Can be stored for a long time e Growth in soft agar: Propagates well (soft agar: 0M160 medium with 0.3% agar and 10% fetal bovine serum adjusted to pH 7.2) f. Serum Requirements: 0M16 with 10% fetal bovine serum
Grows well in 0 medium. Can be grown in 0M160 medium containing fetal bovine serum of Section 2. In the present invention, the method for culturing human hepatoma white rice cell lines capable of producing EPO is as follows.

The medium can contain any nutrient source available to the EPO-producing cells. Specifically, a synthetic medium containing, for example, about 1 to 25 hours of serum can be used. As the serum, newborn bovine serum, fetal bovine serum, horse serum, etc. can be used, and fetal bovine serum is suitable for bamboo. In addition, as synthetic media, DM160, DM170 (manufactured by Kyokuto Seika Kogyo), Nomu F-10, Norm F-12 (manufactured by Hinaga Pharmaceutical), RPM11640 (manufactured by GIBCO), etc. can be used, especially DM16o. is suitable. In addition to serum and synthetic media, these media also contain streptomycin (~100 μg/ml) and penicillin (~100 μg/ml).
00 U/ml), kanamycin (50 μg/ml
It is preferable to add a small amount of antibiotics such as ).

The culture process is basically the same as that of normal cell culture, and in order to produce a large amount of EPO in the cell culture, it is effective to culture under a relatively low acid purple partial pressure. Specifically, for example, EPOi living cells were added to the culture medium for 10'
Add ~166 Cell Is/ml and culture under the following conditions.

Culture gas phase: CO2 = 3-10 qb Air = 90-97% pH near, 0-7.5 Temperature: 37°C In addition, in the above, the oxygen concentration in the culture gas phase is changed by replacing a part of the air with nitrogen, etc. By replacing with gas other than oxygen,
If the amount is reduced to 10% or less, it is possible to increase the production amount of EPO several times. In addition, phenylhydrazine, CAMP, tetradecanoylphorbol r phosphoric acid (TP) were added to the medium.
By adding inducers such as A), EP
It is also possible to measure the amount of O produced.

After the start of culture, the culture medium (culture solution) is replaced with a new one every 2 to 4 days and culture is continued. Once the cells have grown over the entire surface of the culture vessel, treat these cells with a suitable dispersant, for example trypsin (0.02%) to redisperse them, and then repeat the same procedure to start the culture. Chestnut return (
subculture).

Since EPO is produced in culture fluids during the growth phase, these culture fluids can be collected and treated with known purification and separation methods, such as salting out, dialysis, filtration, centrifugation, straightening, freeze drying, etc. It can be easily purified, separated, and collected. In order to further purify EPO, known methods such as adsorption/deactivation on an ion exchanger, gel filtration, affinity chromatography, equal point separation, and electrophoresis may be used in combination as necessary. (For a specific method, see T., Mivake et al., , J.
, Bjol, Chem,, 252(15) 55
58 (1977), S. Lee-Hung 1 (lo
od 56(4) 620 (1980)-, dispute
tl” if it has been done).

Examples 1 to 4 huH-2 cells were suspended in a medium with a composition not shown in the first coating,
Culture was performed under the conditions shown in the left column of Table 2. In addition, as for the cells, lj1 cells were treated with trizocin (
0,'02%) and dispersed therein. Furthermore, the medium was replaced at 31st and 61st days after the start of culture, and m9 was cultured for 29 days.

Table 1 Medium composition (in 1 liter) DM160: 900 ml Fetal bovine blood: 100 ml Kanamycin: 50 mg The culture solutions obtained for each example (3rd issue) were combined into one, and the EPO activity of each was determined. was measured using the FMLC/9Fe method.The measurement results are shown in the right column of Table 2.
Measurement of PO activity is T,! ';aito et T1
1 ; Br1tish J, of llaemato
Loiy, 54.53-58 (1983).

Applicant's agent Kiyoshi Inomata, -rizokunet tlT, t-E I': (7) formula) 1932
August 9, 2019, Mr. Manabu Shiga, Commissioner of the Japan Patent Office 1 Indication of the date of the event 1988 Patent Application No. 71832 2 Title of the invention ■ Process for manufacturing Rithroboedin 3 Relationship with the person making the amendment Case Patent applicant Foundation Cancer Study Group (1 idiot) 4th Deputy Director July 11, 1982 (Mei S 8 Contents of amendment As per the engraving and attached sheet in the Aki I mark that was originally attached to the application (no changes to the content)

Claims (1)

[Scope of Claims] 1. A strain of erythropoietin derived from human liver cancer, which is characterized by culturing Yltl cells in an appropriate medium and producing erythroboetin in the culture. Manufacturing method. The method according to item 1, wherein the cell line is two cell lines or the hu H-2 strain. 3. The method according to claim 1 or 8g2, wherein the culture is carried out under low partial pressure.