JP2648611B2 - Production of human tissue-type plasminogen activator - Google Patents

Description

The present invention relates to a method for producing tissue-type plasminogen activator (hereinafter abbreviated as tPA) produced by normal human cells.

tPA is produced and secreted from vascular endothelial cells and various tissue cells, dissolves fibrin, the main body of thrombus, and is effective as a therapeutic agent for thrombosis.

[Conventional technology]

It is known that tPA includes single-stranded and double-stranded tPA. These thrombolytic activities are double-stranded ones.
Conventionally, what is called tPA, which is larger than the main chain, has been developed in the form of a double strand alone or a mixture of a double strand and a single strand.

Double-chain tPA has a high thrombolytic activity, is not a thrombus that requires a fibrinolytic effect, and has a very high possibility of activating plasminogen in the bloodstream, and has a clinical tendency to bleed. High (JP-A-59-118717).

However, single-stranded tPA, which is considered a precursor of double-stranded tPA, has a higher affinity for fibrin and is converted to double-stranded tPA at a rapid rate as soon as it is adsorbed to fibrin.

Therefore, single-chain tPA can exert plasminogen activity maximally in the blood clot.

Thus, single-chain tPA, whose thrombolytic activity has been relatively inactive, is considered not to act in the bloodstream, and is in a situation where it has been much desired clinically.
There is a strong demand for a method for efficiently producing only main chain tPA.

[Problems to be solved by the invention]

However, when single-chain tPA is produced using cells, one cell is produced during production by the protease contained in the medium or the protease produced by the cells themselves (these proteases are mainly considered to be plasmin or trypsin). There is a problem that it is difficult to convert a chain into a double strand and obtain a single strand efficiently.

Therefore, as a solution to such a problem,
The following methods are known.

That is, a method of culturing or post-treatment in the presence of aprotinin (European Patent Publication No. 41776), a method of using a soybean-derived trypsin inhibitor or aprotinin in a production cell culture solution (Japanese Patent Application Laid-Open No. 59-1979). 11871
7), a method of culturing or inducing production in a medium supplemented with aprotinin or benzamidine (Japanese Patent Application Laid-Open No. 61-1948)
6), a method of producing only one chain by adding aprotinin and 6-aminocaproic acid during purification (Biochem. Biophy
s. Acta 1982, 719 (2), 318-328) and the like are known.

Also, especially when using adherent cells, it is a major problem that cells are detached from the vessel wall or beads during culture.How to prevent this, removing plasmin from serum or adding aprotinin Reported (Kauffman Molecular and Cellular
Biology 5, 1750).

The aprotinin used in these known methods is extremely expensive, and the method of removing plasmin from serum requires complicated operations, and has many problems in industrial practice.

[Means for solving the problem]

The present inventor has proposed tPA which solves the above-mentioned problems of the known art.
After careful examination of the production method, cells, especially adherent cells,
In tPA production, adding p-aminomethylbenzoic acids to the culture medium was found to be extremely effective in improving single-stranded productivity and preventing detachment of cells from the vessel wall and beads, and completed the present invention. did.

That is, the present invention relates to a method for producing human tissue-type plasminogen activator using cells, comprising the step of adding p-aminomethylbenzoic acid to a culture medium. Factor manufacturing method,
And a method for producing human tissue-type plasminogen activator using cells, comprising the steps of:
A method for producing a human tissue-type plasminogen activator, comprising adding aminomethylbenzoic acids.

Examples of the p-aminomethylbenzoic acids used in the present invention include p-aminomethylbenzoic acid, 3-methoxy-4-aminomethylbenzoic acid, and 3-ethoxy-4-
Aminomethylbenzoic acid, 3-hydroxy-4-aminomethylbenzoic acid, 3-fluoro-4-aminomethylbenzoic acid, 3-chloro-4-aminomethylbenzoic acid, 3-methyl-4-aminomethylbenzoic acid, 2 -Amino-4-aminomethylbenzoic acid and the like.

It goes without saying that the p-aminomethylbenzoic acids in the present invention can be used in the form of their ester compounds and hydrochlorides.

In the present invention, the amount of p-aminomethylbenzoic acid added to the medium is 10 ^-14 to 10 ^-1 M, more preferably 10 ^-3 to 10 ^-2.
M.

Examples of the tPA-producing cell used in the present invention include, for example, a DNA sequence encoding a human normal cell-derived human tissue-type plasminogen activator connected to a human-derived metallothionein promoter, a portion of a BPV-derived plasmid and pBR322. Part of plasmid and DN required for transcription termination
Plasmid constructed by incorporating the A sequence
HT-382, a tPA-producing strain obtained by transforming -127 cells.
Strains and the like can be used (JP-A-62-126978).

Further, for example, a plasmid comprising a DNA sequence encoding the human normal cell-derived human tissue type plasminogen activator and a DNA sequence encoding the SV-40 early promoter and a DNA sequence encoding dihydrofolate reductase,
(Chinese hamster ovary) cells, and tPA-producing strains obtained by selecting cells whose genes have been amplified in a medium containing mesotroxate, SV-21-M2.5K7 strain, and the like can be used (Japanese Patent Application Laid-Open No. Sho 62). -126978).

Of course, a combination of means such as mutation and adaptation,
Alternatively, any cell such as a cell transformed with a virus or the like can be used as long as it is a tPA-producing strain.

The culture medium used was Dulbecco's modified Eagle MEM medium (Nippon Pharmaceutical), 199 medium (Nissui Pharmaceutical), Eagle's minimum essential medium (Nissui Pharmaceutical), etc. (Gibco) 0-10% is added, but the component concentration and the like can be changed as necessary.

Further, a third component such as a surfactant may be added as necessary.

Zinc, cadmium or a salt thereof is added as a substance that induces the production of tPA. The addition amount is 1 to 100 μM.

The culture method is not particularly limited, and for example, is performed by the following method.

That is, a roux flask is charged with medium and, if necessary, a tPA inducer, inoculated with an appropriate amount of cells, and grown in a carbon dioxide incubator at an appropriate temperature and an appropriate time, and at the same time as tPA.
Do production.

Alternatively, load the medium into a roux flask, inoculate the appropriate amount of cells, grow in a carbon dioxide incubator at the appropriate temperature and for an appropriate time, and after reaching confluence, switch to the production medium, and again in the carbon dioxide incubator,
Timely production of tPA.

For example, when a 75 cm ² roux flask is used, cells are inoculated at 0.5 to 2.0 × 10 ⁵ cells / ml, and grown at 37 ° C. for 3 to 4 days, and at the same time, tPA is produced.

Alternatively, for example, when a 75 cm ² roux flask is used, the cells are inoculated at 1-2 × 10 ⁵ cells / ml, and the cells are inoculated at 37 ° C., 3-4
Grown for 37 days at 37 ° C, 1-3
Perform tPA production for a day.

According to such a method, the amount of tPA produced was analyzed to be 5 to 10 mg for single-chain tPA / 0 to 2 mg / for double-chain tPA,
The ratio of single strand to the whole was 95% or more.

(Effect of the present invention)

In the present invention, the culture medium or production medium to which conventionally expensive aprotinin or the like has been added is improved by adding inexpensive p-methylbenzoic acids to improve the productivity of single-chain tPA and increase the cell volume. Peeling from walls and beads can be prevented.

〔Example〕

 Hereinafter, the present invention will be described with reference to examples.

Example 1 Cells used for production of tPA were obtained by using a human metallothionein as a promoter, a part of a BPV-derived plasmid and pBR32
HT-382 strain obtained by transforming mouse C-127 cells with a plasmid constructed by incorporating a part of the plasmid, a DNA sequence necessary for transcription termination, and the like was used.

A 75 cm ² roux flask was charged with 20 ml of Dulbecco's modified Eagle's MEM medium supplemented with 10% pre-inactivated fetal bovine serum, zinc chloride was adjusted to 10 μM, and each substance listed in Table 1 was added to each. Create a medium added at a concentration,
Each of the above cells was inoculated at 1.0 × 10 ⁵ cells / ml.

Incubate at 37 ° C for 4 days in a carbon dioxide incubator,
When the confluence (cell number 10 × 10 ⁵ cells / ml) was reached, the concentrations of single- and double-stranded tPA in the culture solution were quantified by the following analysis method, and the results shown in Table 1 were obtained.

The method for analyzing single-stranded and double-stranded tPA in the medium is described below.

I. ELISA method Single-chain ELISA plate (96 well, Corning)
Monoclonal antibody against PA (PAM-1 American Diagonotica), monoclonal antibody against single-chain + double-chain tPA (PAM-2 American Diagonotica) was diluted to 10 μg / ml with a coating solution, and Add 50μ to each well of the plate, leave at room temperature for 2 hours,
Discard the liquid in ll.

After washing with a washing solution, each well is filled with a blocking solution and left at room temperature for 30 minutes or more.

A sample and a standard (0, 1, 2, 4, 8 ng / ml) diluted 1000 to 2000 times are added to each well by 50 μl, and the mixture is left for 2 hours.

After washing with a washing solution, an anti-tPA rabbit antibody is added.

After washing with washing solution, Goat anti Rabbit IgG, Alkaline Ph
Dilute osphatase Conjugate (Sigma) 500 times to each
Add 50 μl to each well and leave for 1 hour.

After washing with the washing solution, the substrate solution (P-Nitrophenyl phosph
ate Sigma 0.6mg /) is added to each well by 50μ, and 30
Leave for a minute.

50 μl of 3NNaOH is added to each well to stop the enzyme reaction.

The absorbance at 405 nm is read with a commercially available ELISA READER.

A calibration curve is created from the standard, and tPA in the sample is
Measure the concentration.

Calculation method Single-stranded tPA amount = PAM-1 measured value (mg /) Double-stranded tPA amount = PAM-2-PAM-1 measured value (mg /) II. Qualitative analysis by immunoblot Laemmuli, with or without reduction of protein by treatment with electrophoresis sample treatment with or without ethanol
Perform electrophoresis by the method of (1970).

The electrophoresed protein is electrically transferred onto nitrocellulose by the method of Towbin et al. (1979).

Nonspecific protein adsorption sites are saturated with bovine serum albumin.

The nitrocellulose is treated with the first antibody, an anti-tPA antibody (eg, a polyclonal antibody from goat or rabbit, or a monoclonal antibody from mouse), and reacted with tPA on nitrocellulose.

After reacting the secondary antibody with the anti-tPA antibody, the antibody is reacted with an antibody against the secondary antibody conjugated to alkaline phosphatase.

As a substrate for alkaline phosphatase, color is developed using bromochloroindolyl phosphate dissolved in a solution containing nitro blue tetrazolium,
Detects the position of tPA or its degradation products, aggregates, etc.

For single-chain tPA, a major band of about 70 KD is obtained regardless of the reduction and non-reduction pretreatment. In the case of non-reduced double-stranded tPA, a major band of about 70 KD is obtained as in the case of single strand, but when reduced, the band of about 70 KD disappears, and bands of about 30 KD and 40 KD are obtained instead. . Approximately 70 KD in the immunoblot of these reduced and non-reduced samples,
By comparing the bands of about 30 and about 40 KD, it is possible to qualitatively estimate the amount ratio of single-stranded and double-stranded in the sample.

Analysis by Immuno Blot is qualitative, but always ELISA
It was used to confirm that there was no inconsistency with the quantitative Data obtained by.

Example 2 Using the same cells as in Example 1, 20 ml of a 75 cm ² roux flask containing 10% of previously inactivated fetal bovine serum added to a Dulbed modified Eagle MEM medium was added.
The cells were inoculated so as to be × 10 ⁵ cells / ml.

Incubate at 37 ° C for 4 days in a carbon dioxide incubator,
When the confluent (cell count 10 × 10 ⁵ cells / ml) is reached, the medium is discarded, zinc chloride having the same composition is adjusted to 10 μm, and each substance listed in Table 2 is added at each concentration. To
Add 20ml each at 37 ℃ in a carbon dioxide gas incubator, 2
TPA was produced for a day, and the tPA concentration in the production medium at that time was analyzed in the same manner as in the Example, and the results shown in Table 2 were obtained.

Example 3 Cells used for the production of tPA were CHO (Chinese hamster ovary) using a plasmid consisting of a DNA sequence encoding the tPA-encoding DNA sequence linked to the SV-40 early promoter and a DNA sequence encoding dihydrofolate reductase. ) The cells were transformed, and the SV-21-M2.5K7 strain obtained by selecting cells whose genes were amplified in a medium containing mesotroxate was used.

Medium similar to that in Example 1 (without adding zinc chloride)
In the same manner, and the results shown in Table 3 were obtained.

Claims (2)

(57) [Claims] 1. A method for producing a human tissue-type plasminogen activator using cells, comprising the steps of:
A method for producing human tissue-type plasminogen activator, comprising adding aminomethylbenzoic acids. 2. A method for producing a human tissue-type plasminogen activator using cells, comprising the steps of: after culturing, adding p-aminomethylbenzoic acid to a production medium containing a human tissue-type plasminogen activator inducer; A method for producing a human tissue-type plasminogen activator, characterized by adding.