JPH0569507B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for assisting the dissolution of tissue plasminogen activator, and more particularly, to a method for assisting the dissolution of tissue plasminogen activator during storage or during operations such as separation and purification, freeze-thaw, freeze-drying, etc. It is something. To date, as plasminogen activators, urokinase isolated and purified from urine or cultured kidney cells, and streptokinase obtained from streptococci have been put into practical use as thrombolytic agents. However, since these have no affinity for fibrin, large doses are often administered in order to obtain the desired effect 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 binds to plasmin inhibitors in the blood and is rapidly deactivated. Therefore, in order to increase the therapeutic effect, it is necessary to administer large amounts of these plasmin to increase the blood circulation. It is necessary to generate plasmin in excess of the amount of plasmin inhibitor. 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 the fibrin, small amounts of fibrin can be degraded without being affected by plasmin inhibitors in the circulating blood, and fibrinogen in the circulating blood can be degraded. The decomposition effect is also weakened. 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. Such plasminogen activators include tissues present in human or animal tissues such as the uterus, kidney, lung, small intestine, foreskin, and blood vessel walls, normal cell cultures derived from these tissues, or tumor cell cultures. It has attracted attention as a plasminogen activator and is expected to be developed as a thrombolytic agent. The tissue plasminogen activator used in the present invention can be extracted and purified from human or animal-derived tissues, or tissue culture fluids derived from these tissues. Examples include cells capable of producing tissue plasminogen activators, such as human fetal kidney, intestine, lung, heart, ureter, skin, foreskin, and normal diploid cells from whole fetuses; Placenta-derived cells or human kidneys, intestines,
Tissue culture fluid using normal diploid cells derived from lung, thyroid, heart, ureter, skin, human melanoma cells or tumor cells with similar properties, particularly preferably derived from human fetal kidney, lung and foreskin. A tissue culture solution using normal diploid cells of 20% is processed using methods commonly used in protein chemistry, such as carrier adsorption, ion exchange, fractional precipitation, gel filtration, electrophoresis, and various affinities. Chromatography, particularly a method using specific antibodies, is desirable, and these methods can be used alone or in combination. Examples of such purification methods include fibrin sepharose column chromatography using fibrin-bound sepharose, CM sepharose column chromatography using carboxymethyl group-bound sepharose, and CM sepharose column chromatography using lysine-bound sepharose. lysine sepharose column chromatography used, ligand exchange chromatography using zinc chelate sepharose, lectin column chromatography using sepharose bound to concanavalin A, and specific plasminogen activator used in the present invention. Antibody affinity chromatography using antibodies that specifically bind to dextran, and gel filtration using crosslinked dextran particles. An example of a specific separation and purification method for the plasminogen activator used in the present invention is as follows:
Add ammonium sulfate to tissue culture fluid or tissue extract, collect the resulting precipitate, dissolve it in rhodanammonium solution containing sodium chloride, pass through an anti-urokinase Ig-G Sepharose column, and adsorb onto a lysine Sepharose column. let
The eluate obtained by using ε-aminocaproic acid as an elution solvent was further added to anti-urokinase Ig.
-G After passing through a Sepharose column, it is freeze-dried and concentrated. The desired plasminogen activator is obtained by gel-filtering the concentrated solution using Sephacryl S-200 (registered trademark of Pharmacia). Note that the titer was measured by the following method (the same applies to the following experiments). Measurement was carried out using an agar-added fibrin plate prepared from 95% coagulated fibrinogen (plasminogen content: approximately 50 casein units/g coagulated protein) using a plate method using urokinase as a standard. The plasminogen activator solution used in the present invention was diluted with 0.067M Tris-HCl buffer (PH8.0) containing 1% gelatin, 0.1M sodium chloride, and 0.1% sodium nitride, and plated on a fibrin plate at 10 IU/ml. The concentration of the plasminogen activator solution used in the present invention, which has the same solubility window as urokinase, was 10 U/ml. The present inventors have been studying the purification of the thus obtained tissue plasminogen activator with high fibrin affinity in order to put it into practical use. , its solubility was found to be significantly reduced. For example, purity 20000U/mg
An aqueous solution containing the protein tissue plasminogen activator (dissolved in physiological saline) is
It does not show solubility higher than 3000U/ml. Under these circumstances, despite its usefulness, it is extremely difficult to efficiently and stably provide the highly purified plasminogen activator on an industrial scale. In order to solve such problems, the present inventors
As a result of extensive research, it was discovered that the solubility of the plasminogen activator was significantly increased by adding lysine to the aqueous solution of the plasminogen activator, and the present invention was completed. be. It is an object of the present invention to provide a method for assisted dissolution of tissue plasminogen activator. This objective is easily achieved by the coexistence of tissue plasminogen activator and lysine. The solubility of the purified tissue plasminogen activator obtained in each of the purification steps described above decreases as the degree of purification increases, but by adding lysine according to the present invention, highly purified tissue plasminogen activator can be obtained. Highly concentrated solutions of plasminogen activator can be obtained. That is, lysine is added in each step of the above-mentioned ion exchange resin, gel filtration, affinity ligand binding resin chromatography, salting out, dialysis, concentration, virucidal heat treatment, filtration, freeze-thaw, freeze-drying, etc. However, if the operation is performed with increased solubility, the objective can be achieved. The lysine used in the present invention may be in the D-form, L-form or racemic form, and may also be in the form of an acid addition salt thereof, such as a hydrochloride,
In addition, commercially available products can be used as they are, but when used for injection, it is desirable to use products of a quality that can be used as raw materials for pharmaceuticals. The amount of lysine that increases the solubility of the tissue plasminogen activator aqueous solution is effective within the saturation solubility of 1 mM or more, preferably 5 mM or more. Practically speaking, the amount of lysine per 3000 to 20000 U of the plasminogen activator is
A concentration of 100mM or less is sufficient, but a concentration higher than that is also acceptable. The pH of the tissue plasminogen activator aqueous solution containing lysine is in the range of 3 to 11, more preferably in the range of 6 to 11, and is preferably maintained by buffering with sodium phosphate or the like.
Salts such as sodium chloride and sodium sulfate
At a concentration of 0.02M to 2M, more preferably 0.5M to
It is preferable to allow them to coexist within a range of 2M. The solution containing the plasminogen activator of the present invention to which lysine has been added is 0 to 30°C, preferably 0 to 10°C, in the solution state.
It is preferable to store it or perform operations such as separation and purification, concentration, and freeze-drying. In a solution containing the plasminogen activator of the present invention to which lysine is added, the solubility of the plasminogen activator of the present invention can be improved even during solution state or cryopreservation, or during operations such as separation and purification and lyophilization.・The activity of the activator is maintained. Next, the present invention will be described in more detail with reference to examples. Example 1 Human fetal lung cells were placed in a 12 volume spinner flask.
They were implanted at a density of 10 ⁵ cells/ml with a concentration of 2.5 mg/ml Cytodex (bead carrier for cell culture, registered trademark of Pharmacia), and grown at 37°C in air containing 5% carbon dioxide with 10% growth medium. Add 8 volumes of medium MEM containing fetal bovine serum, and culture in suspension while stirring at a rotation speed of 40 rpm. After culturing for 8 days and allowing the cells to proliferate sufficiently, wash the bead carrier to which the cells have adhered with physiological saline and remove serum.
Medium 199 with 0.5% Lactalbumin Hydrolyzate
8, and culture while stirring at a rotation speed of 60 rpm. Every fifth day, the culture medium is exchanged and the culture solution containing the plasminogen activator used in the present invention is collected. The culture solution 32 thus obtained is adsorbed onto a CM Sepharose column (2.5φ x 10cm) equilibrated with 20mM acetate buffer (PH4.0) containing 0.1% Twin 80 and 0.15M NaCl. After washing with 20 mM acetate buffer (PH 4.0) containing 0.1% Twin 80 and 0.15 M NaCl, elution was performed with 20 mM Tris-HCl buffer (PH 8.9) containing 0.1% Twin 80 and 1 M NaCl. Solution of plasminogen and moiety with activator activity used
Collect 204ml. Add this solution to 0.1M Rodankali,
0.1% Twin 80 Dialyzed overnight at 4℃ against 20mM Tris-HCl buffer 20 containing 0.05M NaCl and adsorbed onto a lysine sepafrose column (3.6φ x 20cm) equilibrated with the same buffer. After washing with equilibrated buffer, 10mM lysine, 1M rhodankali, 0.2M ε-amino-n-caproic acid and
Elute with 20mM Tris-HCl buffer containing 0.1% Twin80. 251 ml of this eluate was concentrated to 30 ml using a hollow fiber for ultrafiltration, and a Sephacryl S-200 column (5.0 φ
Gel filtration was performed at 92 cm). Collect 78 ml of the solution of the part having plasminogen activator activity used in the present invention. The concentration of the obtained plasminogen activator solution is
It showed a specific activity of 31000U/mg protein at 9200U/ml. Experimental Example 2 The tissue plasminogen activator obtained in Experimental Example 1 was diluted with physiological saline to a concentration of 2000 U/ml, and 30 ml of this was diluted with various lysine-containing buffers.
After dialysis overnight at °C, each was concentrated 10 times using a hollow fiber for ultrafiltration. Table 1 shows the measurement results of solubility and activity after concentration. In addition, the activity of the supernatant of the sample in which aggregates were generated was measured.

[Table] * +: Aggregates present -: Aggregates absent Example 3 Tissue plasminogen activator with a purity of 28,500 U/mg protein was purified from cell culture fluid derived from human fetal foreskin in the same manner as in Example 1. , diluted with physiological saline to 2000U/ml, and 30ml of it.
was dialyzed overnight at 4°C against 20mM phosphate buffer (PH7.5, containing 0.5M sodium sulfate) supplemented with D-form, L-form, or DL mixture of lysine, and then each was dialyzed using a hollow fiber for ultrafiltration. Concentrated 10 times. The results of measuring solubility activity in the same manner as in Example 2 are shown in the second example.
Shown in the table.

【table】

[Table] Example 4 1M NaCl and
Tissue plasminogen activator solution derived from human fetal kidney normal diploid cells dissolved in 20mM phosphate buffer (PH7.5) containing 10mM lysine hydrochloride was concentrated to 20,000U/ml using a limit-overload hollow fiber. 5 ml was dispensed into vials, and the solubility and activity after cryopreservation and freeze-drying storage were measured. The freeze-drying conditions were a freezing temperature of -70°C, a shelf temperature of 20°C, and a drying time of 10 hours. Frozen products and freeze-dried products were stored at -20°C, and freeze-dried products were dissolved in distilled water and measured. The results are shown in Table 3.

【table】

Claims (1)

[Claims] 1. A method for assisting the dissolution of tissue plasminogen activator, which comprises adding lysine to an aqueous solution of tissue plasminogen activator. 2. The lysis assisted method according to claim 1, wherein the tissue plasminogen activator is a plasminogen activator collected from a tissue culture solution of human normal tissue-derived cells.