JPH0558709B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to a method for enhancing the production of a plasminogen activator precursor (hereinafter referred to as a precursor). More specifically, the present invention relates to a method for enhancing production of a precursor, which comprises containing a neutral amino acid and an organic acid or a salt thereof in a medium when producing a precursor by culturing precursor producing cells. [Prior Art] The precursor of the present invention was previously discovered by the present inventor, and its details are described in Japanese Patent Application No. 170354/1982. That is, this precursor is inactive as it is, but it is a type of so-called zymogen that expresses enzymatic activity when treated with plasmin. It has recently been found that this precursor can be produced in human kidney cells in serum-free medium. This precursor has a chain structure of 411 amino acids and has a molecular weight of 5.
However, it shows specific affinity for fibrin and has properties completely different from conventional urokinase. Furthermore, no activity was observed in the synthetic substrate method, but activity was observed in the plate method. Regarding its production conditions, please refer to the patent application filed in 1983.
Although it is described in detail in No.-170354, the production efficiency cannot necessarily be said to be sufficient. [Problems to be Solved by the Invention] Therefore, an object of the present invention is to provide a method for enhancing production of a precursor. [Means for Solving the Problems] Therefore, in order to solve the above object, the present inventors conducted intensive studies on enhancing production of precursors, and found that neutral amino acids and The present invention was completed based on the discovery that the production of precursors can be improved by containing organic acids or their salts at high concentrations. That is, in the present invention, when producing a plasminogen activator precursor by culturing plasminogen activator precursor producing cells, 0.16 to 3.06 w/v% of neutral amino acids and organic acids or The present invention relates to a method for enhancing production of a plasminogen activator precursor, characterized in that the salt thereof is contained in an amount of 0.10 to 3.00 w/v%. (1) Preparation of precursor-producing cells Human kidney cells are preferably used as a raw material for precursor-producing cells. For example, cells that produce precursors obtained by subculturing primary culture or dipioid cells obtained from human kidney cells are used. Inject 200,000 to 300,000 cells/ml of this type of cells,
medium (e.g. waymouth medium or
After continuing culturing for 2-3 days using Dulbecco's modified MEM medium supplemented with 2-5% w/v of heat-inactivated fetal bovine serum, if necessary, replace the medium with a new one and subculture. , cell number 1 million to 2 million
Adjust to cells/ml. (2) Culture conditions As a basic medium, for example, Waymouth medium or Dulbecco's modified MEM medium with 0.05~
A serum-free medium supplemented with 0.2% w/v human serum albumin is used. A neutral amino acid and an organic acid or a salt thereof, which are characteristics of the present invention, are added to this. Examples of neutral amino acids include aliphatic amino acids such as glycine, alanine, leucine, isoleucine, and phenylalanine, but glycine is preferably used. An example of the amount added is 0.1 to 3 w/v%. This is because if the amount added is less than 0.1%, the productivity will not increase sufficiently, and if it is more than 3%, the toxicity will increase, which will have a negative effect on improving the productivity. In addition, the ratio of neutral amino acids to organic acids or their salts is as follows:
A ratio of about 1:10 to 10:1 (weight ratio) is desirable. The organic acid is not particularly limited as long as it has 3 to 18 carbon atoms. Usually, carboxylic acids, especially saturated or unsaturated aliphatic carboxylic acids (especially monocarboxylic acids or dicarboxylic acids) are preferably used. The organic acid may have, for example, a hydroxyl group as a substituent. Examples of such organic acids include fumaric acid, succinic acid, malic acid, citric acid, stearic acid, oleic acid, linoleic acid, palmitic acid, linolenic acid, myristic acid, and the like. Particular preference is given to using aliphatic dicarboxylic acids such as succinic acid. As the salt of the organic acid, physiologically acceptable salts such as alkali metal salts (sodium salts, potassium salts, etc.), alkaline earth metal salts (calcium salts, etc.), etc. can be used. The amount of these organic acids or salts added is exemplified in the range of 0.1 to 3 w/v%. In addition, known additives such as hormones such as lactalbumin hydrolyzate, transferrin, and insulin may be added. For example, 500,000 to 2 million precursor-producing cells are cultured.
The cells/ml are maintained constant in the above medium to produce precursors, and the culture medium is replaced every 2-3 days. The precursors present in this replaced medium are then recovered. Recovery of the precursor from the medium may be performed using known means, such as centrifugation, vacuum concentration, salting out fractionation, gel filtration, concentration, ion exchange chromatography, and affinity chromatography. , by convenient combinations. [Effects of the Invention] According to the method of the present invention, the production of the present precursor can be enhanced by about 1.3 to 3 times in terms of activity ratio compared to conventional methods. Experimental Example 1 In order to examine the effect of enhancing precursor production according to the present invention, an experiment was conducted to compare the production amount of precursors. As shown in Table 1, the amounts of glycine and succinic acid added were set in the range of 0 to 5.0 w/v%. The other conditions were the same as in Example 1 for each group.
Activity was measured as an indicator of production amount. The amount of activity was measured by the p-MCA method as follows. That is, after mixing 0.1 ml of the specimen and 0.1 ml of plasmin solution (0.2 cu/ml, prepared with gelatin buffer [PH8.6]), incubation at 37°C for 10 minutes, and p-MCA solution (0.1 mMGlu-
Add 1 ml of Gly-Arg-MCA) and further heat for 20 minutes at 37°C.
After incubating for minutes and stopping the reaction by adding 18v/v% acetic acid, Ex (excitation wavelength) 370nm,
The fluorescence intensity was measured at Em (fluorescence wavelength) of 460 nm.
The results are shown in Table 1. Note that the unit used was U/ml. U is the international unit of urokinase, and 1U/ml is the fibrinolytic activity expressed by plasmin treatment per ml of sample.
It means equivalent to 1U. Experimental Example 2 Similar to Experimental Example 1, the effects of combined use of the additives listed in Table 2 were investigated, and the results are shown in Table 2. Example 1 A primary culture obtained from human kidney cells was cultured, and only cells producing the precursor of the present invention were isolated and re-cultured, and 5w of heat-inactivated fetal bovine serum was added at a concentration of 20×10 ⁴ cells/ml. /v% containing Waymouth medium, and after 3 days of culture, the number of cells decreased to 100x.
10 ⁴ cells/ml, further increase 0.1w/v
% human serum albumin, 0.5w/v% glycine,
Waymouth containing 0.5w/v% sodium succinate
Add the above precursor producing cells 100x to the medium (serum-free medium)
The cells were replanted at 10 ⁴ cells/ml and cultured for 3 days while maintaining this concentration. The culture solution was centrifuged, fresh medium was added to the precipitate, and culture was continued. The supernatant contained the precursor (activity equivalent to 300 U/ml). The precursor obtained according to the present invention is
It had exactly the same properties as the "fibrinolytic enzyme precursor" disclosed in No. 170354. Example 2 The culture supernatant containing the precursor obtained in Example 1 was adjusted to pH 5.5 and then brought into contact with CM-Sephade×C-50. The adsorbed precursor was eluted with 0.16M phosphate buffer (PH8.5). On the other hand, mice pre-immunized with the precursor
Among the hybridomas obtained by fusing BALB/c spleen cells and mouse myeloma cells with polyethylene glycol, clones with high antibody production based on the precursor were selected. A precursor monoclonal antibody was recovered from the culture solution of this fused cell. This monoclonal antibody was transferred to CNBr-activated Sepharose.
4B (Pharmacia). This monoclonal antibody column was washed with 0.4M NaCl.
The solution was equilibrated with a 0.1M phosphate buffer (PH7.0), and the eluate containing the precursor was contacted therewith.
After washing the column with 0.1M phosphate buffer (PH7.0) containing 0.4M NaCl, remove the adsorbed precursor.
0.2M glycine-HCl aqueous solution containing 0.5M NaCl (PH
2.5). After sterilizing and filtering the eluate,
A highly purified precursor with a specific activity of at least 80000 U/mg was obtained by lyophilization. This purified product showed one band with a molecular weight of 50,000 by SDS-polyacrylamide gel electrophoresis.

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Claims (1)

[Claims] 1. When culturing plasminogen activator precursor producing cells to produce plasminogen activator precursor, add 0.16 to 3.06 w/v% of neutral amino acids and 0.10% of organic acid or its salt to the medium. A method for enhancing production of a plasminogen activator precursor, characterized in that it contains ~3.00w/v%.