JPS60174727A - Stabilization of novel plasminogen activator - Google Patents

Abstract

PURPOSE:To improve the stability of a plasminogen activator in the treatment such as the storage, separation, purification, freeze-drying, etc., and to keep its activity, by adding albumin to a novel plasminogen activator collected from the tissue culture liquid of the cell originated from the normal human tissue. CONSTITUTION:A cell capable of producing plasminogen activator, preferably the cell of lung, kidney and sheath of human fetus, is cultured in various culture liquids, and a novel plasminogen activator is separated from the resultant tissue culture liquid. The plasminogen activator has the following characteristics: molecular weight, 63,000+ or -10,000; isoelectric point, 7.0-8.5; having affinity with fibrin and concanavalin A; optimum pH, 7-9.5; inert to the anti-urokinase specific antibody; etc. The activator can be stabilized by the addition of albumin. The amount of the albumin is selected to give the activator-containing solution having an albumin content of >=1mg, preferably >=5mg per 1ml of the solution. The thrombolytic activity of the activator is 30 times stronger than that of urokinase.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a novel method for stabilizing plasminogen activator, specifically, a novel method for stabilizing plasminogen activator during storage or during operations such as separation and purification, freeze-drying, etc. This relates to a method for stabilizing.

The novel plasminogen activator used in the present invention refers to human normal tissue-derived cells, such as human fetal kidney, intestine, lung, heart, ureter, skin, foreskin, and whole fetus, in a suitable growth medium. cells derived from human placenta, or tissue culture fluid using cells derived from human kidney, intestine, lung, thyroid, heart, ureter, skin, etc., particularly preferably from human fetal kidney, lung, and foreskin. The tissue culture solution using the cells of In particular, it is a substance that has been highly purified using methods using specific antibodies alone or in combination, and has the following properties.

a) Molecular weight: 63,000 ± 10,000 b) Isoelectric point nearness, 0 to 8.5 C) Affinity for fibrin: Yes d) Affinity for concanavalin A: Yes e) Optimal p
H: 7-9.5 f) does not react with anti-urokinase-specific antibodies g) has a specific activity as defined in the text of at least 30,000 units/m Urokinase purified from cells and streptokinase collected from streptococcus have been put to practical use as thrombolytic agents.

However, since these are inferior in their affinity for fibrin, large doses are often administered in order to obtain the necessary effects during treatment, and it is known that side effects such as internal bleeding occur. In other words, the plasmin produced in the circulating blood by these drugs is quickly deactivated by binding to the plasmin inhibitor in the blood, so in order to increase the therapeutic effect, large amounts of these should be administered to inhibit the plasmin inhibitor in the blood. - It is necessary to generate more gold than Blasminge. However, when a large amount of plasmin is produced, it degrades fibrinogen and causes the side effect of bleeding tendency. On the other hand, if fibrin has a high affinity and plasmin can be generated on fibrin, fibrin can be degraded in small amounts without being affected by plasmin inhibitors in the circulating blood, and fibrinogen gold in the circulating blood can be degraded. The decomposition effect also becomes weaker. Under these circumstances, there is a demand for a thrombolytic agent that has high fibrin affinity, is used in small amounts, has high thrombolytic activity, and has few side effects.

The present inventor discovered a novel plasminogen activator gold in a normal cell culture medium and has been considering purifying it for practical use, but it was found that the activity decreases significantly as it is purified to a higher degree. did. Under these circumstances, despite its usefulness, it is impossible to efficiently and stably provide a highly purified new plasminogen activator on an industrial scale.

Thromb Haemost
as).

According to Vol. 48, No. 6, pp. 294-296, 1982, when human serum albumin was added to tissue plasminogen activator derived from melanoma cell culture and the stability was investigated, it was found that it had no effect. has been reported.

As a result of extensive research into the stabilization of a new plasminogen activator, the present inventor found that adding albumin makes it possible to stabilize the plasminogen activator even after long-term storage and after operations such as separation and purification and freeze-drying. found that the activity of the novel plasminogen activator was retained. The present invention is based on this knowledge.

The tissue culture medium of human normal tissue-derived cells used in the present invention is
Cells capable of producing plasminogen activator, particularly preferably human fetal lung, kidney, and foreskin cells, cultured in a spindle culture solution can be used. 54-107510, JP 54-107511, JP 55-19001
No. 4) Kaisho 55-15'? Examples include culture solutions described in Japanese Patent Publication No. 525 and Japanese Patent Publication No. 57-5159. Typical culture methods are illustrated in Reference Examples 1 and 2.

To give an example of a specific separation and purification method for the plasminogen activator used in the present invention, ammonium sulfate is added to a tissue culture medium or concentrated culture medium, the resulting precipitate is fractionated, and sodium chloride is added to the Rodan ammo 1.
The sample is dissolved in a lysine sepharose solution, passed through an anti-throkinase IgG Sepharose column, and adsorbed onto a lysine Sepharose column. This was eluted using ε-aminocaproic acid as a total elution solvent, and the resulting eluate was further added to the anti-urokinase Ig.
-G After passing through a Sepharose column, it is lyophilized and concentrated.

The desired plasminogen activator can be obtained by gel filtration using Sephadex G-150 (registered trademark of Pharmacia). It is clear that it is a plasminogen activator because it completely dissolves fibrin containing plasminogen.

The physicochemical properties of the plasminogen activator used in the present invention thus obtained will be explained below. Note that the titer was measured by the following method (the same applies to the following experiments).

The measurement was carried out using an agar-added fibrin plate prepared from 95% coagulation of ibrinogen (plasminogen content: approximately 50 casein units/V coagulation protein) using a plate method using urokinase as a standard.

Plasminogen activator solution F&K for use in the present invention was diluted with 0.067 M Tris-HCl buffer (pH 8.0) containing 1% gelatin, 0.1 M sodium chloride, and 0.1% sodium nitride. The a degree of the plasminogen activator bath solution used in the present invention was set to 10 1 J/ml, showing the same fusion window as 10 IU/d of urokinase on a fibrin plate.

a) Molecular weight: 63,000±10,0001.5M
HA (Chick Trium, 0.1 M KDT
A, 0. IM Arginine and 0.1-Twin 80 (
0.01 M including Chemical Atlas registered trademark)! Sephadex Q- equilibrated with /phosphate buffer (pH 7,0)
It was measured by gel filtration method using 150'i.

b) Isoelectric point nearness, θ~8.5 The isoelectric point was fractionated and measured by the isofocus electrophoresis method using ampholite.

C) Affinity for fibrin: The plasminogen activator used in the present invention has
When a fibrinogen solution containing no plasminogen was added and then a thrombin solution was added to form fibrin, about 70 units of the plasminogen activator used in the present invention were incorporated into the fibrin. On the other hand, tissue culture urokinase was not incorporated at all.

d) Affinity for concanavalin A: Plasminogen acteve fi-(3otr/+v) used in the present invention
) 2 Go was dissolved in physiological saline and applied to a column (0.5X) of concanavalin A-Sepharose (Pharmacia!
4 cm) and washed with 1M sodium chloride solution, Habo 100- was adsorbed.

e) Optimum pH: 7-9.5 50 μt of plasminogen activator used in the present invention dissolved in physiological saline and 8 CU/ml of plasminogen dissolved in physiological saline containing 10% glycerin.
) 0.05 M citrate buffer (p,) l containing 50 tit and OjO,M sodium chloride 5,0
.

6.0), phosphate buffer (pH 6.0, 7.0, 8.0)
) or glycine-sodium hydroxide buffer (pH 8,
0°9.0, 10.0, 11.0) (pH5,
0, 6, 0, 7, 0.

8.0, 9.0, 10.0, 11.0) were mixed in 100 μt portions and incubated at 37C for 30 minutes. Then 0.1 sM)'JS hydrochloric acid buffer (
fc Boc -Glu -Ly dissolved at pH 8,0)
After adding s-Lys-MCA'i500 ttt and further incubating at 37U for 15 minutes, the reaction was stopped by adding 1-acetic acid, and the produced amine methylcoumarin was measured by fluorescence method to determine the optimum pH. . The measurement results are shown in Figure 1.

f) Reactivity with anti-urokinase specific antibody: JWWtsuov
-su-ase (specific activity 1so, ooo xU/ln9 protein)
After immunizing rabbits with Freund's complete adjuvant for 55 days at 7-day intervals, the blood was collected and purified.
The anti-urokinase specific antibody solution of μr/rnt and the 20 U/mx plasminogen activator solution used in the present invention were mixed at a ratio of 1:1, and the activity of the mixed solution was determined by the method described above. However, no decrease in activity was observed at all.

On the other hand, the urokinase activity of a mixed solution of 20 IU/1 nt urokinase solution and the anti-urokinase antibody solution, which was added as a comparison control, was 100% inhibited.

As described above, the plasminogen activator used in the present invention does not react with anti-urokinase antibodies.

g) The specific activity of two proteins was determined by the method of Lowry et al. (J, Biol, C
hem,.

193, p. 265 (1951)].

The activity was measured by the agar-fibrin plate method described above. According to the above results, the specific activity of the plasminogen activator used in the present invention was at least so, o o units/~protein.

The concentration (IC,) of the protease inhibitor that inhibits the activity of plasminogen activator and urokinase used in the present invention by 50% was determined, and the results are shown in Table 1.

Furthermore, diisopropylfluorophosphate (DFP) K, which is an inhibitor of serine protease, plasminogen activator and urokinase used in the present invention were completely inhibited.

Table 1 (1) KIU (2) Ono Pharmaceutical Co., Ltd. Based on the above properties, the plasminogen activator used in the present invention is a novel urokinase that is different from human urine or renal cell tissue culture-derived urokinase. It is a substance.

Next, the thrombolytic activity of the plasminogen activator used in the present invention was measured using the Chandra loop method (Quar).
t, J, Exp, Physiol, 46, 1 (1
961)). The thrombolysis rate compared to urokinase is shown in Figure 2. Incidentally, fresh human blood was used, and the blood clot formation time was 30 minutes, and the blood clot dissolution time was 4 hours. Furthermore, in FIG. 2, 1 indicates the plasminogen activator used in the present invention, and 2 indicates m-kinase.

As a result, it was confirmed that the thrombolytic activity of the plasminogen activator used in the present invention was 30 times stronger than that of urokinase.

Next, the albumin used in the present invention includes, for example, bovine albumin, toalpmin, and oval albumin, and although there is not much difference in the stabilizing effect of any of them, when used as an injectable preparation, Most preferred is human albumin.

The concentration of albumin added is the plasminogen of the present invention.
The ratio per unit of solution containing the activator is from 90.1 to more than 1, preferably from 1 to more, and even more preferably from 5 to more. The upper limit of the additive concentration is determined from common sense and economical viewpoints, and is 50 Da per 1 ton of the same solution. The amount of albumin to be added to the plasminogen activator gold-containing powder of the present invention is selected so that the above solution concentration is achieved when the powder is dissolved.

Addition methods include directly adding the albumin powder to the plasminogen activator-containing solution of the present invention, dissolving the albumin powder in water or an appropriate buffer before adding it, and adding the same powder to the plasminogen activator-containing solution of the present invention. Minogen・
Examples include a method of adding by mixing with an activator-containing powder, but the method of addition is not particularly limited. The addition time may be during the separation and purification process or during the layering process.

The solution containing the plasminogen activator of the present invention to which albumin has been added is preferably stored at θ~50C1, preferably 0~10C, or subjected to separation, purification, and formulation operations in its solution state. Also,
When storing the same solution in a frozen state, it is desirable to keep it below OC, preferably below -20C. In the solution containing the plasminogen activator added with albumin according to the present invention, the activity of the plasminogen activator of the present invention can be maintained even during storage in a solution state or frozen state, or during separation and purification and formulation operations. is retained.

Next, the present invention will be explained in more detail with reference to Examples.

Reference Example 1 Human embryonic kidney cells 11001111 were implanted at a density of 7 x 10' cells/- in a plastic container, and a medium MgM containing 10 x fetal bovine serum was added as a growth medium in air containing 37C, 5% carbon dioxide gas. After adding fiber and allowing sufficient growth, wash with physiological saline and add serum-free 0.5 thylactalbumin thawed product and 0.8
Production medium 2 consisting of Medium 199 containing % fumaric acid
Change to 0-. After holding for 7 days, the culture medium is replaced with fresh production medium and the culture medium containing the plasminogen activator used in the invention is recovered.

Reference Example 2 Human fetal lung cells f500 2.5 mg/- at a density of 10' cells/- in a spinner flask.
Implanted with mg concentration of Sidedex I (bead carrier for atomic cell culture, registered trademark of Pharmacia), 37C
, 1500 tons of medium MEM' containing 100% fetal bovine serum was added as a growth medium in air containing 5% carbon dioxide gas, and suspension culture was carried out with stirring at a rotation speed of 60 rpm.

After culturing for 8 days and allowing the cells to proliferate sufficiently, the bead carrier to which the cells had adhered was washed with physiological saline, and the bead carrier was washed with serum-free 0.
．． Medium 199 containing 5-thilactalbumin thawed product
300 rpm, and culture with stirring at 60 rpm. While replacing this medium every 5th day,
A culture solution containing the plasminogen activator used in the present invention is collected.

Example 1 10 tons of human fetal lung tissue culture fluid was adsorbed onto a CM Sepharose column (1.5φ x 10crn) equilibrated with 20mM acetate buffer (pH 4.0) containing 0.1% Twin 80 and 0.15M sodium chloride. . 20 containing 0.1 Chituin 80 and 0.15M Sodium Chloride
After washing with mM acetate buffer (p) (4,0), elution was carried out with Zoma) Lis-HCl buffer (pH 8,9) containing 0.1% Twin 80 and 1M sodium chloride to prepare the plasminose used in the present invention. A solution 98- of the moiety with gene activator activity is collected. This solution f O,I
M Rodankali, 0.1 80. 0.05 M sodium chloride in 2 o mM) Lis-HCl buffer 5 t
A lysine Sepharose column (2,6φ
rn) After attaching KM and washing with equilibrated buffer,
0.05 M Sodium Chloride, 1 M Rodankali, 0.2
Elute with 20 mM sodium chloride buffer containing M ε-amino-n-caproic acid and 0.1 titine 80. This eluate was concentrated to 10 ml using a 120 mef hollow fiber, and then transferred to a Sephacryl S-200 column (2.6φX92
Gel filtration was performed using Cn1). A solution 37- of the part having plasminogen activator activity used in the present invention is recovered. The concentration of the plasminogen activator solution obtained was 52 at 4500 U/me.
Using the novel plasminogen activator obtained according to the method of Example 1, the plasminogen activator exhibiting a specific activity of 1200 U/mg protein and having an activity of 1200 U/-・Activator solution (0
, 0.1 MIJ (pH 7.0) containing 15 M sodium chloride was prepared. Various concentrations of human blood W? are added to the plasminogen activator solution. Add albumin and leave at 25C for hr time (3rd, 5th, 10th day)
The residual activity was measured by the activity measuring method described herein, and the residual activity rate (Q) was calculated.

The results are shown in Table 2.

Example 3 A novel plasminogen activator solution purified to 33,000 U/mg from the culture medium of normal diploid + fJII cells derived from human fetal foreskin (0.1 M phosphate buffer containing 0.15 M sodium chloride, pH 7) , 0) was prepared;
Various concentrations of human serum albumin were added to the plasminogen activator solution having an activity of 1200 U/-, and after freezing at -70C, freeze-drying was performed in a lyophilization manner, and then the freeze-dried product was kept at room temperature for one week. After being left to stand, sterile distilled water was added to dissolve it, and the residual activity was measured. The results are shown in Table 3.

Table 3 Example 4 A novel plasminogen activator solution derived from human fetal kidney having the same active concentration as in Example 2 was supplemented with other well-known solution stabilizers for conventional physiologically active substances. Additives were added, the mixture was stored at 25C, and the residual activity rate after 10 days of storage was measured and compared.

The results are shown in Table 4.

Table 4

[Brief explanation of drawings]

Figure 1 is a graph showing the optimal pH range of the plasminogen activator used in the present invention, and Figure 2 shows the relationship between urokinase and the plasminogen activator used in the present invention and thrombolysis rate. The graph shown is Ruru. Figure 1 Figure 2

Claims (1)

[Claims] Adding albumin to a plasminogen activator having the following properties, collected from a tissue culture medium of human normal tissue-derived cells. Activator stabilization methods. a) Molecular weight: 63,000 ± 10,000 b) Isoelectric point nearness, 0 to 8.5 C) Affinity for fibrin d) Affinity for concanavalin A: A) Optimal p
H: 7-9.5 f) Does not react with anti-urokinase specific antibody. g) Specific activity as defined in the text
ity ) is at least so, o o o units/m
9 protein