JP2548562B2 - Manufacturing method of anticoagulant - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for industrially producing an anticoagulant, and more particularly to a method for separating and collecting human tissue-derived cells from tissue culture cells.

[Conventional technology]

The present inventors have succeeded in separating and collecting a novel anticoagulant substance from human placenta, and previously applied for a patent (Japanese Patent Application No.
60-217512).

In this method, placental homogenate is prepared from human placenta, the precipitate is extracted with a buffer solution containing a chelating agent and / or a surfactant, and the extract is subjected to ammonium sulfate fractionation and a known separation / purification method. Blood coagulation substances (hereinafter "PCI")
Abbreviated as “).

[Problems to be solved by the invention]

However, this method is (1) PCI in the raw material placenta
The content is very small, (2) it is difficult to stably supply and secure a large amount of raw material, (3) the hygienic control due to virus contamination of the raw material must always be taken into consideration, (4) contamination of cell contents, etc. Since a large amount of substances are mixed in, purification requires multiple steps, and the yield is low, which is not always satisfactory as an industrial production method.

[Means for solving problems]

In such an actual situation, the present inventors have conducted diligent research to overcome the above-mentioned drawbacks, and as a result, the presence of PCI in tissue-cultured cells of human tissue-derived cells, and a high yield with simple operation from now on. The present invention has been completed by finding that PCI can be collected separately.

That is, the present invention is a method of culturing human tissue-derived cells in tissue, and separating and collecting PCI from the culture.

The cells used in the present invention may be any cells of human origin as long as they have the ability to produce PCI. Examples of such cells include human fetal kidney, lung, small intestine, skin and heart-derived cells; human adult kidney, lung, foreskin, placenta and lymphocyte-derived cells.

Tissue culture of such cells is carried out by a method commonly used for culturing animal cells, for example, Tissue Culture Methods and A
pplications (PKKruse and MKPatterson, Academic
Press., 1973).

The cultured cells thus cultured and proliferated can be used in the present invention as they are, but cells from which interferons, interleukins, plasminogen activators and hormones are extracted can also be used.

Separation and collection of PCI from cultured cells is performed, for example, as follows. That is, the cultured cells are treated with a buffer solution containing a chelating agent such as EDTA, EGTA, oxalic acid, citric acid, sodium nitrilotriacetate and phosphoric acid and / or a surfactant such as Triton X-100, Lubrol, SDS and deoxycholic acid. Treat with a buffer solution containing. The treatment is carried out by immersing the cultured cells in a chelating agent and / or surfactant solution, allowing it to stand overnight at 4 to 8 ° C., centrifuging it, and collecting the supernatant to obtain an extract. Furthermore, the cells of the above residue are put into a chelating agent and / or surfactant solution, the cells are disrupted with a homogenizer, etc., and the supernatant is collected by centrifugation and extracted.
By such treatment, the PCI produced in the cell is eluted outside the cell, and an extract containing a high concentration of PCI is obtained.

Then, from this extract, known separation / purification means such as dialysis, ion exchange chromatography, gel filtration,
Adsorption chromatography, hydrophobic chromatography,
PCI can be isolated and purified by isoelectric focusing, column electrophoresis, affinity chromatography using lectins or antibodies, etc., alone or in combination.

The anti-PCI monoclonal antibody used for the above-mentioned affinity chromatography and confirmation of PCI was found, for example, according to the present invention (Japanese Patent Application No. 61-269588).
No.) It is manufactured by the following method. That is, (1) antibody-producing cells are prepared from an animal immunized with PCI as an antigen, (2) another myeloma cell is purified, (3) these cells are fused, and (4) the obtained hybridoma Are selectively proliferated, (5) the antibody-producing hybridoma is searched from the hybridoma, and (6) the desired monoclonal antibody-producing hybridoma is obtained by cloning.

(1) Preparation of Antibody-Producing Cells Preparation of antibody-producing cells may be performed according to a conventional method, such as a method of immunizing an animal with PCI, which is an antigen, and obtaining antibody-producing cells of the animal. As animals, mice,
Rats, rabbits, guinea pigs, sheep, etc. are exemplified,
As the antibody-producing cells, cells isolated from spleen, lymph nodes, peripheral blood and the like are used. As an immunization method, a method in which Freund's complete adjuvant is used in combination is used.

(2) Preparation of myeloma cells Myeloma cells used for cell fusion are not particularly limited.
Although many mammalian cell lines are available, it is preferable to use animal cell lines that are the same as the animal used to prepare the antibody-producing cells. The cell line to be used considers the separation of unfused and fused cells by allowing unfused myeloma cells to survive the selective medium after cell fusion, allowing only hybridomas to grow. Therefore, those having a specific drug resistance are preferable. For example, 8-azaguanine-resistant cells are preferably used because they have a property that they cannot grow in a HAT medium. Specifically, mouse myeloma cell lines PAI, P3-X63-Ag8, P3-X63-Ag8-U1, P3-NSI / 1-Ag
4-1, X63-Ag8-6.5.3., SP2 / 0-Ag14, FO, S194 / 5XXO.BU.
1, MPC11-45.6.TG.1.7 or the like is used.

(3) Cell fusion Cell fusion is usually performed by mixing myeloma cells with antibody-producing cells in a medium such as MEM medium, RPMI 1640 medium, IMDM medium (mixing ratio is usually 1: 4 to 1:10). It is done by doing. As a fusion promoter, polyethylene glycol (PEG) having an average molecular weight of 1,000 to 6,000 can be used. The working concentration of PEG is usually 30-50%.

(4) Selective Proliferation of Hybridoma Cells after cell fusion are appropriately diluted with IMDM medium containing 10% FCS and centrifuged. The precipitate is suspended in a selective medium (for example, HAT medium), inoculated into a 96-well microplate, and then cultured in a 5% carbon dioxide gas culture device. The cells that grow in the selective medium are hybridomas.

(5) Search for antibody-producing hybridomas Searching for antibody-producing hybridomas may be performed according to a conventional method, and is not particularly limited. For example, after collecting the culture medium in which the hybridoma has grown and reacting with PCI, the enzyme,
It can be searched by the reaction with the secondary antibody labeled with a fluorescent substance, a luminescent substance, or the like.

(6) Cloning The cells in the culture well confirmed to contain the antibody-producing hybridoma are cloned by the limiting dilution method or the like to obtain a monoclonal antibody-producing hybridoma.

By the above operation, PCI-specific monoclonal antibody-producing hybridomas PCI-H39, PCI-H46, PCI-H16
7, PCI-H169, PCI-H176, PCI-H180 were obtained. Each of these hybridomas is a novel cell that produces a monoclonal antibody specifically to PCI. Therefore, we tried to deposit these cells at the Institute of Microbial Technology, Institute of Industrial Science and Technology, but they were rejected as 61 minute contribution No. 1415.

A PCI-specific monoclonal antibody is prepared by using the antibody-producing hybridoma obtained above. That is, by culturing the antibody-producing hybridoma in an appropriate medium, a monoclonal antibody can be obtained from the culture supernatant. In addition, in order to produce a large amount of monoclonal antibody, pristane (2,6,10,14-tetramethylpentadecane) was added to an animal of the same species as the myeloma cell origin.
After intraperitoneally administering a mineral oil such as the above, the antibody-producing hybridoma is inoculated to proliferate a large amount of the antibody-producing hybridoma in vivo. According to this method, high concentrations of monoclonal antibodies are produced in the serum and ascites of the inoculated animals. Separation and purification of the monoclonal antibody can be carried out according to the method used for purification of antibody from normal serum.

Thus obtained monoclonal antibody of the present invention, PIC-A39, PC depending on the type of antibody-producing hybridoma used.
I-A46, PCI-A-167, PCI-A169, PCI-A176 and PC
There are 6 types of I-A180.

 The PCI thus obtained has the following properties.

Molecular weight (SDS-polyacrylamide gel electrophoresis, reduced state) 34,000 ± 2,000 Isoelectric point (isoelectric focusing column electrophoresis using ampholite) 4.7 ± 0.1 Stability b Inactivated by heat treatment at 50 ° C for 30 minutes Stable at ~ 10 Stable in plasma at 37 ° C for 30 minutes i Extends calcium re-coagulation time Extends proprothrombin time Extends activated partial thromboplastin time Amino acid analysis Aspartic acid, threonine, serine, glutamic acid , Proline, glycine, alanine, 1 /
2 Presence of cystine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, histidine, lysine and arginine.

 Sugar analysis No presence of sugar was found in the phenol / sulfuric acid test.

〔The invention's effect〕

As described above, according to the method of the present invention, raw material availability is not restricted, and a highly pure PCI high-concentration solution can be obtained. Therefore, PCI can be produced in a high yield by a simple purification operation. This is an industrially excellent method that can be performed.

〔Example〕

 Next, examples will be described.

Example 1 Various cells shown in Table 1 derived from human placenta were treated with 10% FCS.
(Bovine fetal serum) 1 to 3 × 10 ⁶ roller bolts (750 ml, Falcon) were inoculated with DMEM and cultured at 37 ° C. for 90 seconds / rotation for 5 to 7 days. The surface of cells grown in a monolayer
After thoroughly washing with PBS (phosphate buffer), cool to 4 ℃ 2
A physiological saline solution containing 5 mM EDTA was added, and cells were physically detached from the bottle using a rubber and a spatula. When the cells grew into a monolayer, the number of cells was 1-2 × 10 ⁷ per bottle. The cell detachment solution was centrifuged at 3000 rpm for 10 minutes, and the amount of PCI contained in the supernatant (extract 1) was determined using the monoclonal antibody P
It measured by ELISA (enzyme-linked immunosorbent assay) using CI-A46. Further, the same EDTA solution was added to the precipitate (cells), the cells were disrupted at 4 ° C. using a homogenizer, and the PCI amount contained in the centrifugal supernatant (extract II) was measured in the same manner as above.

 The results are shown in Table-1.

Example 2 (i) Preparation of anti-PCI monoclonal antibody column Promocyan-activated Sepharose 4B (0.4 g) was added to 1 mM hydrochloric acid, 0.1%.
The solution was washed successively with M sodium bicarbonate-0.5 M sodium chloride (pH 8.3) buffer solution to purify a coupling buffer solution (1.5 ml) of promocyan-activated sepharose 4B.

A coupling buffer solution (1 ml) of purified monoclonal antibody PCI-A46 2 mg was added thereto, and the mixture was shaken at room temperature for 2 hours and dehydrated with a glass filter. Furthermore, 0.1M Tris-
10 ml of a hydrochloric acid buffer solution (pH 8.0) was added, and the mixture was shaken at room temperature for 2 hours to block the remaining active site.

The obtained antibody-bound Sepharose 4B was added to 0.1M Tris-hydrochloric acid-0.5M sodium chloride buffer solution (pH 8.3), 0.1M acetic acid-
The column was washed alternately with 0.5 M sodium chloride buffer (pH 4.0) three times and equilibrated with 0.1 M Tris-HCl buffer (pH 7.4) to obtain an antibody column.

(Ii) Purification by Affinity Chromatography The antibody column obtained in (i) was adsorbed with PCI through the extract I of the cultured cells obtained in Example 1 and thoroughly washed with the buffer used for equilibration. Then, PCI was eluted with 0.1 M acetic acid-0.5 M sodium chloride buffer (pH 5.0). Table of the results
It is shown in FIG.

The resulting PCI solution (2.4 mg / ml) 100 μ, protease solution 20 μ (protease type I 1.1 unit / ml, trypsin 880 BAEE unit / ml or 20 mM Tris-HCl buffer (pH 7.
4, control)] and 5 mM containing 0.15 M sodium chloride
A mixed solution of Tris-hydrochloric acid buffer solution (pH 7.4) 80 µ was incubated at 37 ° C, 5 µ was sampled after 0, 1, 2 and 3 hours, and the activated partial thromboplastin time (APTT) was measured. Here, APTT is 5μ sample
APTT reagent 20μ, physiological saline 75μ and standard serum 100μ
Was added and incubated at 37 ° C. for 5 minutes, 200 μl of calcium chloride (0.0125M) was added, and the coagulation time was measured.

As a result, as shown in Table a, PCI retained the activity of 74% or more even when treated with either protease type I or trypsin for 3 hours. At this time, the coagulation time of the negative control to which PCI was not added was 52 seconds.

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Claims (2)

(57) [Claims] 1. A method for producing an anticoagulant, which comprises culturing human tissue-derived cells in tissue, and separating and collecting an anticoagulant having the following properties from the cultured cells. Molecular weight (SDS-polyacrylamide gel electrophoresis, reduced state) 34,000 ± 2,000 Isoelectric point (isoelectric focusing column electrophoresis using amphorite) 4.7 ± 0.1 Stability b Deactivated by heating at 50 ° C for 30 minutes Stable at pH 4 to 10 Stable in plasma at 37 ° C for 30 minutes Stable for at least 74% of anticoagulant activity after 3 hours treatment with protease I or trypsin Action Extend calcium re-coagulation time Extend proprothrombin time Activated partial thromboplastin prolongs time Amino acid analysis Amino acid analysis showed aspartic acid, threonine, serine, glutamic acid, proline, glycine, alanine, 1 /
2 Presence of cystine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, histidine, lysine and arginine. Sugar analysis No presence of sugar was found in the phenol / sulfuric acid test. 2. Separately collecting the cultured cells with a surfactant and / or
Alternatively, the method according to claim 1, which is a method of extracting with an extract containing a chelating agent and then isolating from the extract.