JPS62107795A - Production of physiologically active substance and novel serum-free medium - Google Patents

Abstract

PURPOSE:To culture an animal cell in high efficiency and produce a physiologically active substance, by adding a novel protein produced by a myeloleukemia cell strain K562-T1 to a medium in place of serum. CONSTITUTION:Human myeloleukemia cell strain K562-T1 is cultured in a medium. A novel protein substance accumulated in the culture product is separated therefrom and is added to a medium in place of serum. An animal-originated cell (B-lymphocyte cell such as Namalva, LukII, etc., T-lymphocyte cell such as CCRF-CEM, epithelial cell such as KB, myeloma cell such as SK-Ly-18, RPMI-8226, etc., monocyte cell such as HL-60, etc.) is cultured in the above medium to accumulate a physiologically active substance in the culture product.

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 and a serum-free medium used therefor. 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.

BACKGROUND OF THE INVENTION As a method for culturing animal cells using a serum-free medium, the method described in the following literature is known.

Journal of General Virology (J, gen, Virol, ) 44.227-22
9 (1979), RPMI-1640 was used as the basal medium, 1% (W/V) bovine serum albumin, 0.25%
There is a description of a serum-free medium containing methylcellulose, 0.75% Primaton RL (protein decomposition product), and the like. Journal of Tissue Culture Methods (J, 7
1ssueCulture Methods) 8
(4), 167-17N1983), 3 g/ml insulin and 5 μg/ml) were added to the above basal medium.
There is a description of a serum-free medium containing transferrin as a protein component.

A serum-free medium using human cell-derived substances as in the present invention is not known.

Problems and Means to be Solved by the Invention In methods for producing physiologically active substances 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 and type of serum have a great effect on cell yield and substance production. Therefore, the development of a serum-free medium is desired.

As a result of research on serum-free media, the present inventor found that
It has been discovered that animal cells can be efficiently cultured and physiologically active substances can be produced by adding a novel proteinaceous substance produced by 562-Tl to a myeloid leukemia cell line instead of serum, and the present invention completed.

Structure of the Invention The present invention involves culturing animal-derived cells in a medium containing a proteinaceous substance produced by 562-TI in a myeloid leukemia cell line,
Provided is a method for producing a physiologically active substance, which comprises accumulating the physiologically active substance in a culture and collecting the physiologically active substance from the culture.

The present invention also provides a serum-free medium containing the proteinaceous substance.

The substance of the present invention has the following physical and chemical properties.

■) Molecular weight: 20.000±2.000 molecular weight is S
D S (Sodium dodecyl 5ulfa
te)-Polyacrylamide gel electrophoresis method [New Experimental Chemistry Course (20) Biochemistry, (I) p118, Chemical Society of Japan (Maruzensha Publishing)]. Analysis with an acrylamide concentration of 3% on the concentrating gel and 16% on the separating gel showed a single band at a flowing sample volume of 204.

For staining, silver staining reagent "Daiichi" manufactured by Daiichi Chemical Co., Ltd. was used. As a molecular weight marker, lysozyme (M
W14.400), soybean trypsin inhibitor (M
W21.500), carbonic anhydrase (
M W 31.000), ovalbumin (MW 45,
000), bovine serum albumin (MW66, 200)
and phosphorylase B (VW92, 500). All molecular weight markers are BIO-RADL
ab, manufactured by the company.

(2) N-terminal protein primary structure Met-G I n-11e-Ph e-Va I −
Lys-Thr-Leu-Thr-G ] y
-tys-Thr-11e-Thr-Leu-G I
u-Va I-G Iu-Pro-X-Asp-X-I
Le-X-Asn-Val-X-Ala-XJ]e-(
X indicates an unidentified amino acid. ) The amino acid sequence was provided by Applied Biosystems (Ap
Plied Biosystems) 470A sequencer and Spectra Physics
It was determined by a combination with high performance liquid chromatography (manufactured by RA Physics).

(3) Thermal stability: Stable at pH 7, 3, and 50°C for 30 minutes.

The substance of the present invention (lyophilized powder) was added to PBS (-) (NaCl2) at pH 7.3 so that the concentration was 20 times that of the culture supernatant.
8g/j! , KCA'0.2g/LNazHPO<
1.15 g/I! , KH2PO-0.2g/j
2) and heated in a water bath at 50°C for 30 minutes, cell proliferation promoting activity was measured using lymphoblastoid Namalva strain by the following method.

(4) pH stability: Stable to treatment at 4°C and pH 2 to 3.24 hours.

The culture supernatant of K562-T1 strain was cut to a molecular weight of 3, OO
Concentrate 200 times with O holofiber, and reduce this to 0.1
% acetic acid at 4° C. for 24 hours, and the cell proliferation promoting activity of the dialysate was measured using lymphoblastoid Namalva strain by the following method.

(5) Isoelectric point: 6.5±0.5 Isoelectric focusing method is performed using LKB8100 (11 Qml) (
Using a column manufactured by LKB (Sweden), the concentration of ampholyte ampholyte was adjusted to 1.9 to 0.625%, and the pH was adjusted to 3.
~100 scents are used. Cool to 4℃, 900V, 10
After 48 hours of electrophoresis with mA, fractionation is performed using a fraction collector, and the pH and activity of each fraction are measured.

(6) DNA synthesis-promoting activity and cell proliferation-promoting activity DN
A synthesis promotion activity measurement method Test cells 1-5 x 105 cells/ml at RPM l-16
40 medium (Nissui Pharmaceutical Co., Ltd. glutamine (4mM)
, streptomycin (25 g/m!).

Veninlin (25U/ml), Hepes (10mM)
.

Culture was carried out at 37° C. for 2 days in 25 ml of medium supplemented with baking soda (0.01%) and calf serum (10%).

Centrifuge the culture at 800xg for 5 minutes to collect cells,
Suspend in the above medium minus calf serum to a concentration of 10' cells/ml, dispense into a 24-well multi-dish plate, and culture at 37°C for 24 hours, then transfer to fresh medium. The cells were exchanged and cultured at 37°C for 16 hours. Replace with fresh medium again, sample Q, 1ml, medium Q, 9+ml
was added.

After culturing at 37°C for 6 hours, add 0.5μC of tritium thymidine.
The uptake activity was measured using a liquid scintillation counter. The measured value is 2
The average value of multiple experiments is shown.

Cell proliferation promoting activity Test cell line 5-10 x 105 cells/ml at RPM 1-
Glutamine (4mM) in 1640 medium (Tamizu Pharmaceutical Co., Ltd.)
, streptomycin (25 JLg/m+).

Penicillin (25LI/ml), Hepes (10mM
).

The cells were cultured at 37° C. for 48 hours in 75 ml of a medium supplemented with baking soda (0.01%) and calf serum (10%).

The cells were collected by centrifuging the culture at 800Xg for 5 minutes, washed with the above medium without serum, and dispensed into a 24-well multi-dish plate. 1 ml of sample solution was added. 37℃, C
After culturing in an O2 incubator for 3 days, the number of cells was measured using a hemocytometer.

For adhesion-dependent cells, immediately before measurement, add 0.1%
Measurements were made after adding trypsin to suspend the cells.

The activity was expressed as the value obtained by dividing the number of cells with the addition of the sample by the number of cells without the addition of the sample, with the value without the addition of the sample being taken as 100.

The DNA synthesis-promoting activity and cell proliferation-promoting activity of the proteinaceous substances obtained by the above measurement method against various cells are as shown in Table 1. All cells in Table 1 except those marked with * are human-derived cells.

Table 1 B-cell Namalva +
+Luk II +
+RajiND- SSY-1+ + T-cell Molt-3ND
-CCRF-CEM+
+Myeloma 5K-Ly-18+
+RRMI-8226+
+, P3 [11”
ND-Monocyte HL-5
3+ +[1-937-)
-Epithelial cells KB 10
~pHeLa 10
Ordinary fibroblast 3T3L1” -, -NR
K-49° -- CEF'' -- NO indicates unidentified. + indicates activity, - indicates no activity.

The substance of the present invention can be produced as follows.

The proteinaceous substance used in the present invention is obtained by culturing human myeloid leukemia cells with the 562-TI strain in a culture medium and accumulating them in the culture.
It can be produced by collecting from the culture.

K562-TI strain is obtained by adding K562 strain to 10% fetal calf serum.
These are protein-free medium-acclimated cells obtained by sequentially lowering the serum concentration from Ham's FIO medium containing Ham's FIO medium and undergoing a conditioning period of about one year. The K562 strain was approved by the Proceedings of the National Academy of Sciences (
Proc, Natl.

^cad, Sci,), LISA,, 76, 1
293 (1979), Blood,
To, 321 (1975), GANN,
It is a known cell line described in 73°97 (1982), etc., and is a cell line that can generally be handled manually.

As the culture medium, protein-free media such as Ham's FIO medium, Ham's F12 medium (manufactured by Flow Lab), Dulbecco's MEM medium, MEM medium, RPMI-1640 medium (manufactured by Hinaga Pharmaceutical Co., Ltd.), and mixed media thereof are used. . If necessary, appropriate amounts of 0.5 to 5 mM glutamine, antibiotics [penicillin (25 LI/ml), streptomycin (25 g/ml), etc.], 1 sodium bicarbonate (0.01%), etc. may be added to the medium.

For culture, various culture bottles, Nyare, roller bottles,
A spinner flask, jar fermentor, etc. can be used. The culture medium usually has a seed cell density of 5XlO'
xt ~10@thin 11fi/m+, 30~40℃,
When carried out 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, lXl0' cells/+
A seed cell density of ++1, 37t, 2 days of culture produces 300 units/ml of active substance in the culture medium.

The substance of the present invention is collected from the culture as follows. That is, the obtained culture supernatant was freeze-dried.

Ultrafiltration 1 Concentrate using a strongly acidic ion exchange resin. For elution, use a weakly basic buffer or a low concentration alkaline solution.

The concentrated solution contains a large amount of low-molecular salts and impurities, so it is dialyzed with 1% acetic acid. This removes many impure proteins as precipitates. The acetic acid is removed by freeze-drying, and it is further concentrated to produce a crude product. Purified product. Pass the crude product through a cation exchange resin. The active substance has only a weak interaction with the cation exchange resin and is not adsorbed but elutes. This active fraction is collected and freeze-dried. After concentrating with etc., low-molecular substances derived from the medium are removed by gel filtration.Gel filtration agents include Sephadex (manufactured by Pharmacia Fine Chemicals), biogels (manufactured by Bio-Rad), control Boa glass (manufactured by Corning Glass Works), Toyo Pearl (manufactured by Toyo Soda), etc. are used.

When Bio-Get 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 Namalva
.

B-lymphoid cells such as Suk II, CCRF-CE
T-lymphoid cells such as M, non-T non-B lymphoid cells such as K562, epithelial cells such as KB, 5K-Ly-18
, Myeloma lineage cells such as RPMI-8226, H
Examples include Monocyte cells such as L-60.

The physiologically active substances produced by the method of the present invention include interferon-α, -β, -γ, interleukin-I, -
It includes anything that can be produced by animal cells, such as II, -■, TNF, lymphotoxin, monoclonal antibodies such as TgG, and immunosuppressive substances.

In the method of the present invention, the medium used for culturing animal cells includes:
RPMI-1640, MEMSDME as basal medium
(hereinafter manufactured by Tamizu Pharmaceutical Co., Ltd.), Ham FIO, Ham F12, GE
M (manufactured by Flow Lab), DM160, DM170
(all manufactured by Kyokuto Pharmaceutical Industries, Ltd.) and other commonly commercially available media.

The medium contains, if necessary, glutamine 0.5-5mM, penicillin 10-5QU/ml, streptomycif 10-50g/ml, heavy iJO, 05-0.5%,
Hebes 1.0-5QmM, Sodium pyruvate 0.5-5
Appropriate amounts of 0 mL selenite 10-' to 10-''M, galactose 0.1 to 110 1/ml, etc. may be added.

Proteinaceous substances are added to the medium at a concentration of 1-1000 ng/ml.

For culture, various culture bottles, petri dishes, roller bottles,
A spinner flask, jar fermentor, etc. can be used. Culture is usually carried out at a seed cell density of 5 x 10'~
When the culture is carried out at 1X10' cells/ml at 30 to 40°C for 2 to 4 days, physiologically active substances are mainly produced in the culture medium depending on the cell density.

Collect physiologically active substances from the culture as follows. That is, the obtained culture supernatant was subjected to lyophilization, ultrafiltration,
Collect using gel filtration, ion exchange resin, etc.

The serum-free medium of the present invention includes, if necessary, glutamine 0.5-5 mMs Hebes 1-5mM, penicillin 10-50 (J/ml, streptomycin 10
~50 mM, baking soda 0.05-0.5%, sodium pyruvate 0.5-50mM, selenite 10-6-10
-'M, add galactose 0.1-10 mg/ml, etc., and add protein substances produced by myeloid leukemia cells to 5.0% or less, preferably 2.0-0.01% (W/V)
It was added.

Example I RPMI-1640 medium (manufactured by Tamizu Pharmaceutical Co., Ltd.) was used as the basal medium, 4mM glutamine, 10mM Hepes, 25U/ml
Penicillin, 25μ/ml streptomycin, 0.0
1% baking soda and LGF-11% produced in Reference Example 1 (
W/V) in 10 ml of serum-free medium containing 5 x 10' cells/
ml of Namalva cells were suspended and cultured at 37° C. for 2 days in a 25 cri Co-Ning culture flask.

After culturing, add the same serum-free medium to a total volume of 30ml.
The cells were cultured at 37° C. for 2 days in a cm Corning culture flask. After culturing, the same serum-free medium was added to a volume of 90 ml and cultured at 37°C for 2 days in a 25 Qml spinner flask (manufactured by Shibata Vario). 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 7×105 cells/ml.

HVJ (Sendai virus) 50HAU/m to culture
1 of the mixture, and was left at 37°C for 5 hours to adsorb the virus, and then at 28°C for 16 hours to induce interferon. Culture at 5.000 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.

The interferon activity in the supernatant was measured by the above method and the results are shown in Table 2.

Table 2 Example 2 Various cells were cultured using serum-free medium LGF-R1. Dispense 5 x 10' cells/ml of each cell into a 24-well multititer plate and place in a C02 incubator at 37°C for 3 hours.
Cultured for 1 day. The number of cells in the culture solution was measured using a hemocytometer. LGF-1 with medium without LGF-1 added as 1
The growth rate at various concentrations is shown in Table 3.

Table 3 Example 4 Serum-free medium LGF-R1 was prepared by mixing the following substances with the basal medium.
shall be.

Basal medium RPMI-1640 (manufactured by Hinaga Pharmaceutical Co., Ltd.) Nglutamine 4mM Penicillin 2511/ml Streptomycin 25ml/ml Hepes 1QmM Baking soda 0.01% LCF-1
2,0~0. [) 1% (W/V) (manufactured in Reference Example I a)
(Adjust as appropriate depending on the application) Example 5 The following substances were mixed with the basal medium to make serum-free medium LGF-01.
shall be.

Basal medium Darbetzko et al.'s MEM (manufactured by Tamizu Seiyaku Co., Ltd.) Glutamine 4 m M Veninrin 15 U/ml Streptominone 15 μ/ml Baking soda 0.01% LG F
-12,0~0.0125% (W/V) (Adjust as appropriate depending on the application) Reference example 1゜Myeloid leukemia cell line containing 562-T1 strain 8 x 105 cells/
The mixture was placed in a 202 large spinner flask containing 8 p of the following medium at a concentration of 1.0 ml, and cultured at 37° C. for 3 days. The medium is
As a basal medium, Hammu FIO medium (manufactured by Flow Lab)
and Darubetsuko et al.'s MEM medium (manufactured by Hinaga Yakuhin Co., Ltd.).
・Pyruvic acid (5mM), mixed at a ratio of 1:1
Selenite (L, 25 X 10-'M), galactose (1 mg/ml), glutamine (4 mM), penicillin (25 U/ml), streptomycin (25 g
A protein-free medium supplemented with sodium bicarbonate (0.01%) and sodium bicarbonate (0.01%) was used. Using a hollow fiber (molecular weight cut-3000, Amicon Inc.*), culture supernatant 120β was
All the information was added to Qml. Approximately 10 Qml of the concentrate was dialyzed against 1% acetic acid at 4°C for 24 hours. The resulting precipitate was removed by centrifugation (12,00 rpm, 10 minutes), and the supernatant was freeze-dried to remove acetic acid to obtain a powder. Distilled water
Dissolved in 2 Qml.

16 Qml capacity Q A E -3ephadex
-A50 (7y manufactured by Lumacia Fine Chemical Co.)
The solution obtained above was passed through the column in two portions of 60 ml each. Elute with 15 Qml of PBS (-),
Further elution with 0.1% acetic acid 19Qn+l, PBS
300 ffl+ of the active fraction eluted with (-) was obtained. The activity recovery rate was about 8.3%, and the activity increased 1.9 times.

30 Qml of this active fraction was freeze-dried, dissolved in 20ml of distilled water, and passed through a column containing Blo-Ge1 P-6040Qml in two portions at SV5.0. P.B.
Elute with S(-), fractionate the eluate into 5 ml portions,
5 Qml of the ~45th half was obtained. Recovery rate of activity is 1
．． At 7%, the specific activity increased 13.3 times. This active fraction was transferred to FPL manufactured by Falman Fine Chemical Co., Ltd.
C(Fast protect+n 1quid ch
romato) Hraphy ) 14ono S 5
It was passed through a column containing [111]. Washed with PBS(-) and eluted with 0-0.5 M saline.

The eluate was fractionated into 1 [111] fractions, and activity was observed 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 4 The present invention provides a serum-free medium suitable for culturing cells derived from animals, particularly humans. The serum-free medium of the present invention contains human-derived proteins and is free from foreign protein contamination, so it is suitable for culturing human-derived cells, and it is suitable for collecting useful substances from the culture.
4 is also advantageous.

Claims (7)

[Claims] (1) Animal-derived cells are cultured in a medium containing a proteinaceous substance produced by myeloid leukemia cell line K562-T1, physiologically active substances are accumulated in the culture, and the physiologically active substances are collected from the culture. A method for producing a physiologically active substance, characterized by: (2) The method according to claim 1, wherein the proteinaceous substance has the following physical and chemical properties. 1) Molecular weight: 20,000±2,000 (SDS-PA
GE method) 2) N-terminal protein primary structure [amino acid sequence is included] (X indicates an unidentified amino acid.) 3) Thermal stability: Stable when treated at pH 7.3, 50°C, 30 minutes. 4) pH stability: Stable for treatment at 4°C, pH 2-3 for 24 hours 5) Isoelectric point: 6.5±0.5 6) Has a proliferation-promoting effect on the following cells. Human B lymphocyte cell Human T lymphocyte cell Human Myeloma cell Human Monocyte cell Human non-T non-B cell 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 (3) The method according to claim 1, wherein the animal-derived cells are selected from the cells listed in Table 1. (4) The physiologically active substance is interferon-α, interferon-β, interferon-γ, IL-2,
2. A method according to claim 1, characterized in that it is IL-3, CSF or TNF. (5) A serum-free medium containing a proteinaceous substance produced by myeloid leukemia cell line K562-T1. (6) The serum-free medium according to claim 5, wherein the proteinaceous substance has the following physical and chemical properties. 1) Molecular weight: 20.000±2.000 (SDS-PA
GE method) 2) N-terminal protein primary structure [amino acid sequence is included] (X indicates an unidentified amino acid.) 3) Thermal stability: Stable when treated at pH 7.3, 50°C, 30 minutes. 4) pH stability: 4℃, pH 2-3, stable for 24 hours treatment 5) Isoelectric point: 6.5±0.5 (7) The serum-free medium according to claim 5, which contains the proteinaceous substance at a ratio of 5.0% (W/V) or less.