JPH0148759B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a method for producing physiologically active substances using animal cells. The present invention is useful for the pharmaceutical industry because it can be applied to cell culture of interferon, which is useful as an anticancer drug. Prior Art As a method for culturing animal cells using a serum-free medium, the method described in the following document is known. According to Journal of General Virol. 44 , 227-229 (1979):
RPMI-1640 as basal medium, 1% (w/v) bovine serum albumin, 0.25% methyl cellulose, 0.75
There is a description of a serum-free medium containing % Primaton RL (protein decomposition product). Journal of Tissue Culture Methodology
According to Methods) 8 (4), 167-171 (1983),
There is a description of a serum-free medium containing 3 μg/ml insulin and 5 μg/ml transferrin as protein components in the same basic medium. A serum-free medium using human cell-derived substances as in the present invention is not known. Problems to be Solved by the Invention and Means for Solving the Problems In a method for producing a physiologically active substance by culturing animal cells, the culture is usually carried out in the presence of serum in a conventional culture medium. The presence of serum is essential, and the type of serum and difference in lot greatly affect cell yield and substance production. Therefore, the development of a serum-free medium is desired. As a result of research into a serum-free medium, the present inventor discovered that animal cells can be efficiently grown by adding a novel proteinaceous substance produced by the myeloid leukemia cell line K562-T1 to the medium instead of serum. discovered that it is possible to culture and produce physiologically active substances,
The invention has been completed. Structure of the Invention The present invention involves culturing animal-derived cells in a medium containing a proteinaceous substance produced by myeloid leukemia cell line K562-T1, accumulating physiologically active substances in the culture,
A method for producing a physiologically active substance is provided, which comprises collecting the physiologically active substance from the culture. The substance of the present invention has the following physical and chemical properties (1) Molecular weight: 20000±2000 Molecular weight is SDS (Sodium dodecyl sulfate)-polyacrylamide gel electrophoresis [New Experimental Chemistry Course (20) Biochemistry () p118, Nippon Chemical (Maruzensha Publishing)]. Analysis with an acrylamide concentration of 3% on a concentrating gel and 16% on a separating gel showed a single band with a flow sample volume of 20μ. For staining, silver staining reagent "Daiichi" manufactured by Daiichi Chemical Co., Ltd. was used. As a molecular weight marker,
Lysozyme (MW14400), soybean trypsin inhibitor (MW21500), carbonic anhydrase (MW31000), ovalbumin (MW45000), bovine serum albumin (MW66200) and phosphorylase B (MW92500) were used. All molecular weight markers are manufactured by BIO-RAD Lab. (2) N-terminal protein primary structure Met-Gln-Ile-Phe-Val-Lys-Thr-Leu
−Thr−Gly−Lys−Thr−Ile−Thr−Leu−Glu
-Val-Glu-Pro-X-Asp-X-ILe-X-
Asn-Val-X-Ala-X-Ile- (X indicates an unidentified amino acid.) The amino acid sequence was determined using an Applied Biosystems 470A sequencer and Spectra Physics.
Determination was made in combination with high performance liquid chromatography (manufactured by Physics). (3) Thermal stability: Stable for processing at PH7.3, 50℃, 30 minutes. The substance of the present invention (lyophilized powder) was added to PBS (-) (NaCl 8
g/, KCl 0.2g/, Na ₂ HPO ₄ 1.15g/,
KH ₂ PO ₄ 0.2g/) in a water bath at 50°C.
After heating for 30 minutes, cell proliferation promoting activity was measured using lymphoblastoid Namalva strain by the following method. (4) PH stability: Stable for 24 hours at 4℃ and PH 2-3. The culture supernatant of the K562-T1 strain was concentrated 200 times using a holographic fiber with a molecular weight of 3000, and this was dialyzed using 0.1% acetic acid at 4°C for 24 hours to determine the cell proliferation promoting activity of the dialysate. It was measured using the Namalva strain by the following method. (5) Isoelectric point: 6.5±0.5 Isoelectric focusing method is performed using 110ml of LKB8100 (LKB
Using an ampholyte ampholyte column (Sweden), the concentration of ampholyte ampholyte was 1.9 to 0.625%, and the pH was 3 to 10.
Use the slope of Cooled to 4℃, 900V, 10mA
After 48 hours of electrophoresis, fractionate using a fraction collector and measure the pH and activity of each fraction. (6) DNA synthesis promoting activity and cell proliferation promoting activity DNA synthesis promoting activity measurement method 1 to ⁵ x 105 cells/ml of test cells were added to RPMI-1640.
Culture medium (manufactured by Nissui Pharmaceutical Co., Ltd.) contains glutamine 4mM, streptomycin 25μg/ml, penicillin 25U/ml,
The cells were cultured at 37° C. for 2 days in 25 ml of a medium containing 10 mM Hepes, 0.01% sodium bicarbonate, and 10% calf serum. Centrifuge the culture at 800 x g for 5 minutes to collect cells, and
Suspend the cells in the above medium without calf serum to a concentration of 10 ⁵ cells/ml, dispense into a 24-well multi-dish plate, and culture at 37°C for 24 hours.
The medium was replaced with fresh medium and cultured at 37°C for 16 hours. Replace with fresh medium again, sample 0.1ml, medium 0.9ml
was added. After culturing at 37°C for 6 hours, tritiated thymidine was added at 0.5 μCi/well, and its uptake activity was measured using a liquid scintillation counter. The measured values are the average values of two or more experiments. Cell proliferation promoting activity Test cell line 5-10×10 ⁵ cells/ml with RPMI-
4mM glutamine in 1640 medium (Nissui Pharmaceutical Co., Ltd.)
Streptomycin 25μg/ml, penicillin 25U/
ml, Hepes 10mM, baking soda 0.01% and calf serum 10
% and cultured at 37°C for 48 hours. The cells were collected by centrifuging the culture at 800 × g for 5 minutes, washed with the above medium minus serum, aliquoted into a 24-well multi-dish plate, and mixed with 0.9 ml of serum-free medium and 0.1 mL of serum-free medium. ml of sample solution was added.
After culturing for 3 days at 37°C in a CO ₂ incubator, the number of cells was measured using a hemocytometer. For adhesion-dependent cells, immediately before measurement,
Measurement was performed after suspending the cells by adding 0.1% trypsin. The activity is 100 for the sample without additives.
The value is calculated by dividing the number of cells when the sample is added by the number of cells when the sample is not added. The DNA synthesis promoting activity and cell growth promoting activity of the proteinaceous substances obtained by the above measurement method on various cells were as shown in Table 1. All cells in Table 1 except those marked with * are human-derived cells. [Table] [Table] Indicates none.
The substance of the present invention can be produced as follows. The protein substance used in the present invention can be produced by culturing human myeloid leukemia cell line K562-T1 in a medium, allowing it to accumulate in the culture, and collecting it from the culture. The K562-T1 strain is a protein-free medium-acclimated cell obtained by sequentially lowering the serum concentration of the K562 strain from Ham's F10 medium containing 10% fetal calf serum and undergoing an acclimatization period of about 1 year. The K562 stock is owned by the Proceedings of the National Academy of Sciences.
Of Science (Proc.Natl.Acad.Sci.),
USA., 76 , 1293 (1979), Blood,
45, 321 (1975), GANN, 73 , 97 (1982)
This is a commonly available cell line among the known cell lines described in, etc. Media include Ham's F10 medium, Ham's F12 medium (manufactured by Flow Lab), Dulbecco's MEM medium,
Protein-free media such as MEM medium and RPMI-1640 medium (all manufactured by Nissui Pharmaceutical Co., Ltd.) and mixed media thereof are used. Glutamine may be added to the medium if necessary.
Appropriate amounts of 0.5-5mM, antibiotics [penicillin 25U/ml, streptomycin 25μg/ml, etc.], baking soda 0.01%, etc. may be added. For culturing, various culture bottles, shears, roller bottles, spinner flasks, jar fermenters, etc. can be used. The culture medium usually has a seed cell density of 5 × 10 ⁴ × 1 to 10 ⁶ cells/ml, and a seed cell density of 30 to 40
℃ for 2 to 4 days, the substance of the present invention is mainly produced in the culture solution depending on each cell density. For example, 1
At a seed cell density of ×10 ⁵ cells/ml and incubation for 2 days at 37° C., 300 units/ml of active substance are produced in the culture medium. The substance of the present invention is collected from the culture as follows. That is, the obtained culture supernatant is concentrated using freeze-drying, ultrafiltration, strongly acidic ion exchange resin, or the like. For elution, use a weakly basic buffer or a low concentration alkaline solution. Since the concentrated solution contains many low-molecular salts and impurities, it is dialyzed with 1% acetic acid. This removes much of the impure protein as a precipitate. Acetic acid is removed by freeze-drying, and the product is further concentrated to obtain a crude product. The crude product is passed through a cation exchange resin. The active substance has only a weak interaction with the cation exchange resin and is not adsorbed but eluted. The active fractions are collected, concentrated by freeze-drying, etc., and then low-molecular substances derived from the medium are removed by gel filtration. Gel filtration agents include Cephadex (Pharmacia,
(manufactured by Huain Chemical Co., Ltd.), biogels (manufactured by Biorad Co., Ltd.), control pore glass (manufactured by Corning Glass Works Co., Ltd.), Toyo Pearl (manufactured by Toyo Soda Co., Ltd.), etc. are used. When Bio-Gel P-60 or the like is used, the active fraction is located between the void volume (Vo) and low molecular weight substances such as salts. The concentrate obtained by the above operation can be purified as a single component by further performing high performance liquid chromatography. A specific method for producing the proteinaceous substance used in the present invention is shown in Reference Example 1. The animal cells used in the method of the present invention include:
B-lymphocyte cells such as Namalva, Luk, etc.
T-lymphoid cells such as CCRF-CEM, non-T non-B lymphoid cells such as K562, epithelial cells such as KB, Myeloma cells such as SK-Ly-18 and RPMI-8226
Examples include Monocyte cells such as HL-60 and Monocyte cells. The physiologically active substances produced by the method of the present invention include interferon-α, -β, -γ, interleukin-, -, -, TNF, lymphotoxin,
It includes anything that can be produced by animal cells, such as monoclonal antibodies such as IgG and immunosuppressive substances. In the method of the present invention, the media used for culturing animal cells include RPMI-1640, MEM,
DME (hereinafter manufactured by Nissui Pharmaceutical Co., Ltd.), Ham F10, Ham F12,
GEM (manufactured by Flow Lab), DM160, DM170
(all manufactured by Kyokuto Pharmaceutical Industries, Ltd.) and other commonly commercially available media. Glutamine 0.5~
5mM, penicillin 10-50U/ml, streptomycin 10-50μg/ml, baking soda 0.05-0.5%, Hepes
1.0-50mM, sodium pyruvate ^0.5-50mM , selenite ^10-6-10-8 M, galactose 0.1-10
An appropriate amount such as mg/ml may be added. Proteinaceous substances are added to the medium at a concentration of 1-1000 ng/ml. For culturing, various culture bottles, shears, roller bottles, spinner flasks, jar fermenters, etc. can be used. Culture is usually carried out at a seed cell density of 5 x 10 ⁴ to 1 x 10 ⁶ cells/ml, with a seed cell density of 30 to 40 cells/ml.
℃ for 2 to 4 days, physiologically active substances are mainly produced in the culture solution depending on each cell density. Collect physiologically active substances from the culture as follows. That is, the obtained culture supernatant is collected using freeze-drying, ultrafiltration, gel filtration, ion exchange resin, or the like. The serum-free medium of the present invention includes, if necessary, 0.5-5mM glutamine, 1-5mM hepes,
Penicillin 10-50U/ml, streptomycin 10
~50mM, baking soda 0.05-0.5%, sodium pyruvate
Add 0.5-50mM, selenite ^10-6-10-8M , galactose 0.1-10mg/ ^ml , etc., and add proteinaceous substances produced by myeloid leukemia cells to 5.0% or less, preferably 2.0-0.01% (W /V) added. Example 1 RPMI-1640 medium (manufactured by Nissui Pharmaceutical Co., Ltd.) was used as the basal medium, 4mM glutamine, 10mM Hepes, 25U/
ml penicillin, 25μg/ml streptomycin,
LGF-1 manufactured using 0.01% baking soda and Reference Example 1
5x in 10 ml of serum-free medium containing 1% (w/v)
10 ⁵ cells/ml of Namalva cells were suspended and cultured in a 25 cm ² Corning culture flask at 37°C for 2 days. After culturing, add the same serum-free medium to 30 ml.
The cells were cultured in a 75 cm ² Corning culture flask at 37°C for 2 days. After culturing, the same serum-free medium was added to a total volume of 90 ml, and cultured at 37°C for 2 days in a 250 ml spinner flask (manufactured by Shibata Hario). After culturing,
The same serum-free medium was added to make 500 ml, 1 mM sodium butyrate was added, and the culture was further cultured at 37°C for 1 day. The cell density at this time was 7×10 ⁵ cells/
It was hot in ml. HVJ (Sendai virus) 50HAU/
ml and left at 37°C for 5 hours to adsorb the virus, and then at 28°C for 16 hours to induce interferon. culture at 5000 rpm,
The cells were separated by centrifugation for 5 minutes, and 500 ml of the supernatant was placed in a tray and the virus was sterilized using a UV lamp. Table 2 shows the results of measuring the interferon activity in the supernatant using the method described above. [Table] [Table] Example 2 Various cells were cultured using serum-free medium LGF-R1. Dispense 5 x ¹⁰ cells/ml into a 24-well multititer plate and place in a CO ₂ incubator.
The cells were cultured at 37°C for 3 days. The number of cells in the culture solution was measured using a hemocytometer. Table 3 shows the proliferation rate at each concentration of LGF-1, with the LGF-1-free medium being 1. [Table] Example 4 Prepare serum-free medium by mixing the following substances with basal medium.
Let it be LGF-R1. Basal medium RPMI-1640 (manufactured by Nissui Pharmaceutical Co., Ltd.) Glutamine 4mM Penicillin 25U/ml Streptomycin 25μg/ml Hepes 10mM Baking soda 0.01% LGF-1 2.0-0.01% (W/V) (Produced in Reference Example 1) (Depends on the application (adjust as appropriate) Example 5 Mix the following substances with the basal medium to make a serum-free medium.
Let it be LGF-D1. Basal medium Darbetzko et al.'s MEM (manufactured by Nissui Pharmaceutical Co., Ltd.) Glutamine 4mM Penicillin 15U/ml Streptomycin 15μg/ml Baking soda 0.01% LGF-1 2.0-0.0125% (W/V) (Adjust as appropriate depending on the application) Reference example 1 8 x 10 ⁵ myeloid leukemia cell line K562-T1
The cells/ml were placed in 20 large spinner flasks containing 8 of the following medium and cultured at 37°C for 3 days. The culture medium was Ham-F10 medium (manufactured by Flow Lab) and MEM of Darbetsuko et al.
A mixture of culture medium (manufactured by Nissui Pharmaceutical Co., Ltd.) at a ratio of 3:1, pyruvic acid 5mM, selenite 1.25x
10 ^-7 M, galactose 1 mg/ml, glutamine
A protein-free medium containing 4 mM penicillin, 25 U/ml, streptomycin, 25 μg/ml, and 0.01% sodium bicarbonate was used. Approximately 120% of the culture supernatant was filtered using a hollow fiber (molecular weight cut-3000, manufactured by Amicon).
It was concentrated to 100ml. Approximately 100 ml of the concentrate was dialyzed against 1% acetic acid at 4°C for 24 hours. The resulting precipitate was removed by centrifugation (12,000 rpm, 10 minutes), and the supernatant was freeze-dried to remove acetic acid to obtain a powder. Distilled water
Dissolved in 120ml. The solution obtained above was divided into two 60 ml portions and passed through a 160 ml QAE-Sephadex-A50 (manufactured by Pharmacia Huain Chemical Co.) column.
Elute with 150 ml of PBS(-), then add 0.1% acetic acid 190
ml and active fraction eluted with PBS(-) 300
Got ml. The activity recovery rate was approximately 8.3%, and the activity increased 1.9 times. 300 ml of this active fraction was freeze-dried, dissolved in 20 ml of distilled water, and passed through a column containing 400 ml of Bio-Gel P-60 in two portions at SV 5.0. PBS(−)
The eluate was fractionated into 6 ml portions to obtain 60 ml of the 41st to 45th fractions. The recovery rate of activity was 1.7%;
Specific activity increased 13.3 times. This active fraction was analyzed using FPLC (Fast) manufactured by Pharmacia Huain Chemical Co., Ltd.
protein liquid chromatography) Mono S 5
It was passed through a column containing ml. Washed with PBS(-) and eluted with 0-0.5M saline. 1ml of eluate
The activity was found in the 53rd fraction. The recovery rate of activity was 0.2%, and the specific activity increased 3600 times. This active substance is called LGF-1. The results of the above purification process are shown in Table 4. [Table] Effects of the Invention The present invention provides a serum-free medium suitable for culturing cells derived from animals, particularly humans. Since the serum-free medium of the present invention contains human-derived proteins and is free from foreign protein contamination, it is suitable for culturing human-derived cells, and is also advantageous for collecting and purifying useful substances from the culture.

Claims (1)

[Scope of Claims] 1. Animal-derived cells are cultured in a medium containing a proteinaceous substance produced by myeloid leukemia cell line K562-T1 and having the following physicochemical properties, and physiologically active substances are accumulated in the culture. 1. A method for producing a physiologically active substance, the method comprising: culturing the physiologically active substance, and collecting the physiologically active substance from the culture. 1 Molecular weight: 20000±2000 (SDS-PAGE method) 2 N-terminal side protein primary structure Met-Gln-Ile-Phe-Val-Lys-Thr-
Leu−Thr−Gly−Lys−Thr−Ile−Thr−Leu
-Glu-Val-Glu-Pro-X-Asp-X-Ile-
X-Asn-Val-X-Ala-X-Ile- (X represents an unidentified amino acid.) 3. Thermostability: Stable to treatment at PH7.3, 50°C, 30 minutes. 4 PH stability: Stable at 4℃, PH2-3, 24 hour treatment. 5 Isoelectric point: 6.5±0.5 6 Has a proliferation-promoting effect on the following cells. Human B lymphoid cells Human T lymphoid cells Human Myeloma cells Human Monocyte cells Human non-T non-B cells 7 It has a DNA synthesis promoting effect on the following cells. Human B lymphoid cells Human T lymphoid cells Human Myeloma cells Human Monocyte cells Human non-T non-B cells Human epithelial cells