JPS6213928B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing a plasminogen activator with a molecular weight of 45,000 to 68,000 in high yield using cells derived from animals. Plasminogen activators with a molecular weight of 45,000 to 68,000 are known to be useful substances, and the method of isolating and purifying them from human urine is a well-known industrial manufacturing method. However, the conventional method using human urine as a raw material has drawbacks such as unstable quality of the raw material, serious hygiene problems, and difficulty in obtaining large amounts of urine from healthy people. Therefore, as a method to solve these drawbacks, attention has been paid to a method of producing a plasminogen activator using cells derived from animals. This method can supply raw materials of constant quality in large quantities without the risk of contamination, and it is expected that the technology will be industrialized. However, the previously known plasminogen activator produced by cells derived from animals was developed by G.H. Barlow (GH
Barlow, Thrombosis Research, vol. 1, p. 201,
1972, Thrombosis Research, vol. 11, p. 149,
As reported by (1977) et al., it is a mixture of two types, one with a molecular weight of approximately 50,000 and one with a molecular weight of approximately 30,000. The present inventors previously discovered that a plasminogen activator with a molecular weight of 45,000 to 68,000 is produced as a single component by cells derived from the kidney or lung, and further researched methods for its isolation and acquisition. As a result, it was discovered that by treatment with a metal chelating agent in the presence of a metal chelating agent during the separation and acquisition process, the production activating factor can be recovered as it is stably and in high yield without undergoing decomposition or denaturation, and the present invention has been made based on this finding. It came to this. That is, the present invention comprises cells derived from human kidneys or lungs that have the ability to produce plasminogen activator, and a nitrogen source, a carbon source, inorganic salts, and, if necessary, an organic nutrient source. When separating and obtaining a plasminogen activator with a molecular weight of 45,000 to 68,000 from a solution containing the plasminogen activator obtained by contacting the plasminogen with a solution, it is recommended that a metal chelating agent be present during this separation and obtaining step. The present invention relates to a method for producing a plasminogen activator having a characteristic molecular weight of 45,000 to 68,000. First, the production of a plasminogen activator with a molecular weight of 45,000 to 68,000 will be outlined. The cells used in the present invention may be any cells derived from human kidney or lung and have the ability to produce plasminogen activator. These cells can be grown by a culture method commonly used for culturing animal cells, such as the method described in "Tissue Culture" (edited by Junnosuke Nakai et al., Asakura Shoten, 1976). The solution that is brought into contact with the cells in order to cause the cells obtained in this way to produce plasminogen activator is conventionally used to produce plasminogen activator using cells derived from animals. All solutions that have been prepared can be used. Sugars, ie, sucrose, maltose, or glucose, are used as the carbon source contained in this solution. The nitrogen source is preferably an organic nitrogen source, more preferably an amino acid mixture or/and a protein hydrolyzate. The protein used may be either animal or vegetable, but in particular hydrolysates of casein, soybean protein, lactalbumin and meat protein give good results. In addition to the carbon source and nitrogen source, inorganic salts and/or organic additives are added if necessary. Preferred additives in addition to these various nutritional sources include organic acids. Among them, succinic acid,
More preferred are substances selected from malic acid, fumaric acid, and glycolic acid. Even more preferred is fumaric acid. Serine, threonine, or glycine may be added instead of or simultaneously with these organic acids. The pH of these solutions is maintained between 6 and 9, and the temperature is between 15°C and 45°C, more preferably 25°C.
The molecular weight can be reduced by maintaining the temperature between
45,000 to 68,000 plasminogen activators can be produced, and in particular, the contact residence time of these solutions with cells should be 30 days or less (i.e.,
By exchanging these solutions within 30 days), plasminogen activator containing 95% or more of the molecular weight range of 45,000 to 68,000 can be produced for more than 60 days. More preferably, by setting the retention time to 10 days or less (that is, exchanging these solutions within 10 days), the plasminogen activator containing the molecular weight category of 45,000 to 68,000 as a single component can be used for 40 days or more. can be produced in large quantities. Note that the solution may be replaced continuously or discontinuously. The solution obtained in this way has a low concentration of plasminogen activator and contains many impurities in order to be put to practical use. It is necessary to get it separated. As such separation and acquisition steps, adsorption methods, salting-out methods, dialysis methods, chromatography methods, gel filtration methods, electrophoresis methods, etc. are commonly used alone or in combination. These methods are
-10232, JP-A-52-7485, JP-A-53-79092
No. 79093, No. 81686, No. 86083, No. 86084,
There are various disclosures in Nos. 88388, 91184, and 136582, etc., and more specifically, ammonium sulfate,
Salting out method using sodium chloride, sodium sulfate, ammonium chloride, etc., dialysis method, adsorption method using silica gel, hydroxyapatite, barium sulfate, acrylonitrile-methyl acrylate copolymer, etc., ion exchange chromatography using carboxymethyl dextran, phosphocellulose, etc. method, lysine-agarose, benzamidine-agarose, para-aminobenzamidine agarose,
Affinity chromatography using arginine ester-agarose, gel filtration using acrylamide gel, agarose, cross-linked dextran gel, etc., preparative disc electrophoresis using polyacrylamide gel, isoelectric focusing using an amphoteric carrier, and other methods are known. The present invention is characterized in that a metal chelating agent is present in the solution in the separation and acquisition step using these methods alone or in combination. As is clear, it reduces decomposition and denaturation during the separation and acquisition process, and has a high purity and high concentration of homogeneous molecular weight plasminogen activator (molecular weight 45,000).
to 68,000) with high recovery rates. Examples of the metal chelating agent used in the present invention include ammonium N-nitrosophenylhydroxylamine, ammonium perpillate, α-benzoin oxime, 2,3-butanedione dioxime, trans-1,2-diaminocyclohexanetetraacetic acid. , 4,5-dihydroxybenzene-1,3-disulfonic acid, 2,3-dimercapto-1-propanol, diphenylthiocarbazone, 2,2'-dipyridyl, 3,6-disulfo-1,8-dihydroxynaphthalene , dithiooxamide, eriochromeshwarz T, ethylene glycol (2-aminoethyl ether)-N'.
N'tetraacetic acid, ethylenediamine, ethylenediaminetetraacetic acid, ortho-hydroxybenzaldehyde oxime, salicylic acid, 8-hydroxyquinoline, 8-hydroxyquinoline sulfonic acid, 4-methyl-1,2-dimercaptobenzene, 5-nitro-1, 10-phenanthroline, orthophenanthroline, ethylxanthine potassium salt, diethyldithiocarbamyl sodium salt,
Examples thereof include 2-thenoyl-2-furoylmethane, thenoyltrifluoroacetone, and thiourea. These metal chelating agents may be used alone or in combination of two or more, and among them, ethylenediaminetetraacetic acid, trans-1,2-diaminocyclohexanetetraacetic acid, and orthophenanthroline give preferable results. The preferred concentration of the metal chelating agent in the solution is 0.1 millimolar (mM) or more, more preferably 0.3 mM or more. The method of the present invention has the drawbacks of conventional methods such as low concentration of raw urine, difficulty in collecting large amounts of urine of stable quality from healthy people, and hygiene problems in handling urine. , the drawbacks such as low molecular weight purity of the recovered plasminogen activator have been removed, and it has a molecular weight of 45,000 to 68,000,
The present invention is suitable as an industrial method for producing a plasminogen activator, which produces a highly concentrated plasminogen activator of stable quality with high purity and high recovery rate. The plasminogen activator obtained by the method of the present invention has high thrombolytic activity, has uniform component molecular weights, and can be standardized as a substance. Therefore, it is easy to standardize the efficacy as a medicine, and stable administration effects can be expected. Next, the present invention will be explained in more detail with reference to Examples. Example 1 A solution having the composition shown in Table 1 was given to fully grown human kidney-derived cells (Flow Laboratories) and maintained at 37°C. The pH of the solution was adjusted to 7.3, and the dissolved oxygen concentration was adjusted to 50% saturation or higher. The entire solution was replaced every 7 days, and the cells were left in contact with the solution for a total of 49 days. The solution was collected, and its plasminogen activator was measured by fibrin plate method and found to be 628 CTA units/ml, with a molecular weight of 628 CTA units/ml.
It was a single composition of 45,000 to 68,000. This solution
Ammonium sulfate was added to 250 ml to achieve 70% saturation, and the precipitate was collected by centrifugation and dialyzed at 4°C for 48 hours against 10 mM phosphate buffer, pH 7.0, containing 1 mM ethylenediaminetetraacetic acid. Next, this enzyme solution was equilibrated with 10mM phosphate buffer of pH 7.0 containing 1mM ethylenediaminetetraacetic acid to CM Sephadex C50 (Pharmacia).
The solution was adsorbed to the solution and eluted with 10mM phosphate buffer of pH 7.0 containing 0.3M sodium chloride and 1mM ethylenediaminetetraacetic acid, and the solution was further concentrated.
It was purified by a gel filtration method using a 50mM phosphate buffer with a pH of 7.0 containing 0.5M sodium chloride and 1mM ethylenediamine as a developing solvent. The plasminogen activator recovered by this method is
The response rate was 56% with 87,900 CTA units. In addition, molecular weight purity was measured by gel filtration using Cephadex G-100 using cytochrome C, chymotrypsinogen, oalbumin, and bovine serum albumin as molecular weight markers. was obtained alone. On the other hand, when 250 ml of a stock solution containing the same plasminogen activator as above was used as a starting material and purified under the same conditions except that the solution used for post-treatment did not contain ethylenediaminetetraacetic acid, the recovered Plasminogen activator is
The response rate was 23% with 36,100 CTA units. Table 1 Amino acids mg/L L-arginine hydrochloride 21.1 L-cystine 12.0 L-glutamine 292.0 L-histidine hydrochloride monohydrate 10.5 L-isoleucine 26.2 L-leucine 26.2 L-lysine hydrochloride 36.5 L-methionine 7.5 L- Phenylalanine 16.5 L-threonine 23.8 L-tryptophan 4.0 L-tyrosine 18.1 L-valine 23.4 Vitamin D-biotin 1.0 D-Ca-pantothenic acid 1.0 Choline chloride 1.0 Folic acid 1.0 i-inositol 1.8 Nicotinamide 1.0 Pyridoxal hydrochloride 1 .0 Riboflavin 0.1 Thiamine hydrochloride 1.0 Inorganic salt NaCl 8000.0 KCl 400.0 Na ₂ HPO ₄・2H ₂ O 60.0 KH ₂ PO ₄ 60.0 MgSO ₄・7H ₂ O 100.0 CaCl ₂ (anhydrous) 140.0 MgCl ₂・6H ₂ O 100.0 Others Glucose 1000.0 Lacto albumin Hydrolyzate 5000.0 Disodium fumarate 5000.0 Example 2 A solution having the composition shown in Table 1 was given to sufficiently grown human lung-derived cells (Flow Laboratories) and maintained at 37°C. The pH of the solution was adjusted to 7.3, and the dissolved oxygen concentration was adjusted to 52% saturation or higher. The entire solution was replaced every 7 days, and the cells were left in contact with the solution for a total of 42 days. When this solution was collected and the plasminogen activator was measured, it was found to be 158 CTA units/ml, all of which was a single component with a molecular weight of 45,000 to 68,000. 200ml of this enzyme stock solution was fractionated with ammonium sulfate in the same manner as in Example 1, and the precipitate was collected at 4°C with 1mM trans-1.
PH containing 2-diaminocyclohexanetetraacetic acid
6.0 in 10 mM citrate buffer for 48 hours, followed by adsorption onto phosphocellulose equilibrated in the same buffer and subsequent elution with 0.3 M sodium chloride in the same buffer. . Further concentrate this,
0.5M sodium chloride, 1mM trans1.2-
PH8.0 with diaminocyclohexanetetraacetic acid
The product was purified by gel filtration using Sephadex G100 using 0.05M Tris-HCl buffer as a developing solvent. The plasminogen activator thus obtained was 15,200 CTA units, with a recovery rate of 48%. When the molecular weight of this preparation was measured by the same gel filtration method as in Example 1, a single plasminogen activator with a molecular weight of 45,000 to 68,000 was obtained. On the other hand, using 200 ml of the stock solution containing the same plasminogen activator as above as the starting material, purification was performed under the same conditions except that the solution used for post-treatment did not contain trans-1,2-diaminocyclohexanetetraacetic acid. The plasminogen activator recovered was 5690 CTA units, and the recovery rate was
It was 18%. Example 3 3 g of acrylonitrile-methyl acrylate copolymer was added to 200 ml of a solution containing 680 CTA units/ml of plasminogen activator obtained in the same manner as in Example 1, and stirred for 1 hour to prepare plasminogen activator. 4% containing 1mM ortho-phenanthroline at low temperature.
Elution was performed with 50 ml of aqueous ammonia. Subsequently, it was dialyzed for 48 hours against a 10 mM phosphate buffer containing 1 mM ortho-phenanthroline, pH 7.0, and then adsorbed onto hydroxyapatite equilibrated using the same buffer. It was eluted and fractionated while being continuously replaced with a phosphoric acid buffer. The part with plasminogen activation activity was collected and further diluted with 1mM
Gel filtration was carried out using Sephadex G100 using 0.05M phosphate buffer with pH 7.0 containing ortho-phenanthroline and 0.5M sodium chloride as a developing solvent. PH7.0 further containing 1mM ortho-phenanthroline 0.5M sodium chloride,
Plasminogen activator was adsorbed on para-aminobenzamidine agarose equilibrated with 0.05M phosphate buffer, and eluted with 0.1M acetate buffer at pH 4.0 containing 1mM orthophenanthroline and 1M sodium chloride. The total activity of the plasminogen activator thus obtained is
The recovery rate was 31% with 42,400 CTA units. This preparation showed a single band in disk electrophoresis and SDS polyacrylamide gel electrophoresis, and showed that bovine serum albumin, ovalbumin, chymotrypsinogen,
When the molecular weight was measured by SDS polyacrylamide gel electrophoresis using cytochrome C as a standard protein, only a single protein with a molecular weight of 45,000 to 68,000 was observed. On the other hand, when 200 ml of a stock solution containing the same plasminogen activator was used as a starting material and post-treatment was performed under the same conditions except that the solution used for post-treatment did not contain ortho-phenanthroline, plasminogen was recovered. The total activity of the activator is
The response rate was 14% with 19,000 CTA units. Comparative Example 1 A solution containing 97 CTA units/ml of plasminogen activator was obtained using fully grown whole human fetus cells (Flow Laboratories) in the same manner as in Example 1. This plasminogen activator was all a single component with a molecular weight of 45,000 to 68,000. 100 ml of this solution was fractionated with ammonium sulfate in the same manner as in Example 1, and the precipitate was dialyzed at 4° C. for 48 hours against 10 mM phosphate buffer, pH 7.0, containing 1 mM ethylenediaminetetraacetic acid. The total activity after dialysis is
There were 4750 CTA units, and the survival rate was 49%. On the other hand, a precipitate was similarly obtained from 100 ml of the same solution and dialyzed against a buffer containing no ethylenediaminetetraacetic acid, and the residual activity was 52%. As a control, when the solution obtained in Example 1 was treated in exactly the same manner as above, the residual activity was 99% and 42%, respectively. In other words, it has been found that the effect of adding a metal chelating agent is effective for obtaining plasminogen activator from a production solution of cells derived from kidney or lung. It has no effect on all fetal cells to prevent the decomposition, denaturation, and inactivation of the plasminogen activator, and the solution after dialysis in Comparative Example 1 contains plasminogen in the molecular weight category of about 30,000. Contains 62% Gen activator and has a molecular weight of 45,000 to 68,000
It was not possible to directly obtain the plasminogen activator without reducing its molecular weight. Example 4 A solution containing plasminogen activator obtained in the same manner as in Example 1 was fractionated with ammonium sulfate in the same manner as in Example 1, and the precipitate was treated with various concentrations of orthophenanthroline, Table 2 shows the results of dialysis for 48 hours against a 10 mM phosphate buffer with a pH of 7.0 containing trans-1,2-diaminocyclohexanetetraacetic acid and ethylenediaminetetraacetic acid.

[Table] Remarkable effects were observed at addition amounts of 0.1mM or more,
In particular, almost quantitative recovery was achieved when 0.3mM or more was added.

[Brief explanation of the drawing]

The figure shows a gel pattern of Cephaadeix G100, a plasminogen activator obtained by the method of Example 1. The horizontal axis shows the fraction number, and the vertical axis shows the relative activity of the enzyme. Furthermore, the elution position of the standard protein is indicated by an arrow in the figure.

Claims (1)

[Scope of Claims] 1. Cells derived from human kidneys or lungs that have the ability to produce plasminogen activator, and a nitrogen source, carbon source, inorganic salts and, if necessary, an organic nutrient source. When separating and obtaining a plasminogen activator with a molecular weight of 45,000 to 68,000 from a solution containing the plasminogen activator, at least one metal chelating agent is present during the separation and obtaining step. Let the molecular weight be 45,000 or 68,000
A method for producing a physiologically active substance, characterized by obtaining a plasminogen activator at a high concentration and in a high yield. 2. The method for producing a physiologically active substance according to claim 1, wherein the metal chelating agent is ethylenediaminetetraacetic acid, trans-1,2-diaminocyclohexanetetraacetic acid, or orthophenanthroline. 3. The method for producing a physiologically active substance according to claim 1, wherein the concentration of the metal chelating agent is 0.1 mmol or more. 4. The method for producing a physiologically active substance according to claim 3, wherein the concentration of the metal chelating agent is 0.3 mmol or more.