JPH08225461A - Purification of plasminogen and plasminogen preparation - Google Patents

Abstract

PURPOSE: To obtain a purified plasminogen product capable of giving a plasminogen preparation not exhibiting side effects such as an vasodepressor activity in a high yield by adding a cationic surfactant to a plasminogen- containing composition and subsequently recovering a clean solution. CONSTITUTION: The method for purifying the plasminogen comprises adding a cationic surfactant (preferably cetyltrimethylammonium bromide, cetylpyridium chloride) to a plasminogen-containing composition such as a serum or an abdominal dropsy preferably so as to give a concentration of 0.2-0.8w/v% in the composition, and subsequently recovering a clear solution to obtain the objective purified product. The pH of the plasminogen-containing composition of raw material is preferably 6.5-8.5, and the cationic surfactant is preferably added to the above-mentioned composition having a purification degree of approximately 0.005-0.5cu/OD280nm . After the plasminogen-containing solution is treated with the cationic surfactant, the surfactant is preferably removed from the plasminogen-containing solution.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for purifying plasminogen and a plasminogen preparation. More specifically, it relates to a method for purifying plasminogen, which recovers a clear solution containing plasminogen in high yield, and a plasminogen preparation purified by the method.

[0002]

BACKGROUND OF THE INVENTION Plasminogen is activated by urokinase, streptokinase, etc. to become plasmin, which decomposes fibrin to cause a fibrinolysis phenomenon. Therefore, plasminogen, together with urokinase and streptokinase. In addition to the treatment of thrombosis, it is attracting attention as a drug that can be widely applied clinically. Especially that N
The usefulness of lysyl-type plasminogen whose terminal is a lysyl residue is expected.

Conventionally, as the purification method of plasminogen, isoelectric point-acid extraction method, ammonium sulfate fractionation method, polyethylene glycol fractionation method, affinity chromatography method using lysine-agarose, gel filtration method, ion exchange method. A method of treating with the body is known.

Especially the affinity chromatography method,
Purification methods such as gel filtration and ion exchanger treatment can efficiently obtain a highly purified product by column chromatography. However, blood, body fluid, placenta and other biologically-derived components or plasminogen was fractionated and extracted from a culture fluid obtained by a genetic engineering method, and the crudely purified product extracted contains impurities derived from raw materials. Turbidity may be observed in the plasminogen-containing solution obtained by centrifuging or filtering. Alternatively, turbidity may occur due to protein denaturation in the purification step. Such turbidity causes clogging of the column during purification by column chromatography, and there is a problem that the purification purity decreases or the purification operation becomes difficult, especially when a large amount of raw materials are purified. Therefore, a purification method capable of recovering a clear plasminogen-containing liquid without turbidity is desired. Further, a method for purifying plasminogen in a high yield with less loss of plasminogen activity at that time is desirable.

An object of the present invention is to provide a purification method for recovering a clear plasminogen-containing liquid from a plasminogen-containing composition. Another object of the present invention is to provide a method for purifying plasminogen in high yield. A further object of the present invention is to provide a plasminogen preparation which does not show side effects on the circulatory system such as blood pressure lowering effect.

[0006]

Means for Solving the Problems As a result of intensive studies to achieve the above object, the present inventor has found that by using a cationic surfactant, plasminogen can be produced from a plasminogen-containing composition. It was found that the containing clear solution was obtained in high yield. Furthermore, it has been found that plasminogen prepared through the purification method of the present invention has no side effects on the circulatory system such as an antihypertensive effect and can be safely used as a medicine. The present invention has been completed based on such findings.

That is, the present invention is a method for purifying plasminogen, which comprises adding a cationic surfactant to a plasminogen-containing composition and collecting a clear solution. The present invention also relates to a preparation containing plasminogen purified by the purification method. Further, the present invention relates to a plasminogen preparation which has substantially no blood pressure lowering effect.

As the plasminogen which is the target substance in the purification method of the present invention, methionyl type (N-terminal amino acid is methionine), glutamyl type (N-terminal amino acid is glutamic acid) or lysyl type (N-terminal amino acid is Lysine) and the like. Also, a mixture of these may be used. Preferred is lysyl-plasminogen.

The plasminogen-containing composition which is the starting material in the present invention is subjected to the method of the present invention in the state of an aqueous liquid. Plasminogen is present in blood, body fluid, placenta, etc., and the plasminogen-containing composition can be prepared from these by a generally known method.

The plasminogen-containing composition as a starting material is not particularly limited as long as it contains plasminogen, and examples thereof include serum, plasma, ascites, placenta extract, placenta tissue extract. Fractions II + III or III of cold alcohol fractionation of corn from liquid or plasma
Examples include a composition containing a fraction and other fractions obtained by treating plasma or tissue extract with various fractionation methods, a culture solution obtained by culturing a gene recombinant host or tissue culture, and the like.

The plasminogen-containing composition which is the starting material of the present invention preferably has a pH of 5.5 to 9.0, more preferably pH 5.9 to 8.5, and particularly preferably pH 6.
It is 5 to 8.5. When the plasminogen-containing composition does not satisfy the pH, it is preferable to adjust the pH in advance. The adjustment is, for example, Tris buffer,
This is performed with a phosphate buffer solution, a bicarbonate buffer solution, a carbonate buffer solution, or the like. When the pH of the composition is 6.5 to 8.5, the clarification effect is high and the plasminogen activity recovery rate is high.

The degree of purification of the plasminogen-containing composition to which a cationic surfactant is added is not particularly limited, and the method of the present invention can be applied to any degree of purification. Therefore, the addition of cationic surfactant is applied at any stage of the purification of plasminogen. Preferably, 0.
The method of the present invention is applied to a plasminogen-containing composition having a degree of purification of about 005 to 0.5 cu / OD _{280 nm} .

The concentration of plasminogen in the plasminogen-containing composition to which the cationic surfactant is added is not particularly limited, but is, for example, 1 to 100 cu / ml.
The degree is illustrated.

The purification method of the present invention is carried out by adding a cationic surfactant to the plasminogen-containing composition and collecting a clear solution. Specifically, after adding the cationic surfactant, the composition is allowed to stand for an appropriate period of time (eg, 0.
After stirring for 5 to 6 hours, the supernatant is collected by a method such as centrifugation or filtration.

In the method of the present invention, a cationic surfactant is used as the surfactant. Cationic surfactants are excellent in clarifying effect. The cationic surfactant used in the present invention is not particularly limited, and quaternary ammonium salt, alkylpyridinium salt and the like can be used. Examples of quaternary ammonium salts include:
Examples thereof include alkyl trimethyl ammonium salts (usually having 8 to 18 carbon atoms of alkyl) such as cetyl trimethyl ammonium bromide, cetyl trimethyl ammonium chloride, dodecyl trimethyl ammonium bromide and dodecyl trimethyl ammonium chloride, benzalkonium chloride and benzethonium chloride. Examples of the alkylpyridinium salt (usually having 8 to 18 carbon atoms) include cetylpyridinium chloride, cetylpyridinium bromide, dodecylpyridinium chloride and dodecylpyridinium bromide. You may use these cationic surfactants in combination of 2 or more types. More preferably, it is at least one selected from quaternary ammonium salts and alkylpyridinium salts, and particularly preferably at least one selected from cetyltrimethylammonium bromide and cetylpyridinium chloride.

The concentration of the cationic surfactant added is usually 0.1 to 2.0 w / v%, preferably 0.2 to 2.0% in the composition.
1.0 w / v%, more preferably 0.2-0.8 w / v
% Is exemplified. The addition concentration of the cationic surfactant is 0.
When it is 2 to 0.8 w / v%, the clarification effect is high and the activity recovery rate of plasminogen is high.

The treatment with the cationic surfactant may be carried out in the presence of a stabilizer. Examples of the stabilizer include aprotinin, ε-aminocaproic acid (EACA), benzamidines (benzamidine, p-aminobenzamidine, etc.), and basic amino acids (arginine, lysine, etc.). The concentration of stabilizer added is 10 to 10% aprotinin.
About 100 KIE / ml, 1-10 w / v% by EACA
Benzamidines are about 0.0001 to 0.1M, and basic amino acids are about 0.1 to 1M.
The treatment with the cationic surfactant is usually performed at a temperature of 0 to 40 ° C, preferably 2 to 30 ° C.

The purification method of the present invention can be combined with a known plasminogen purification method. Known plasminogen purification methods include, for example, ammonium sulfate fractionation method (JP-A-50-100213), polyethylene glycol fractionation method (JP-A-3-164171), lysine-affinity chromatography (JP-A- Sho 49-1255
No. 06, Science, 170, 1095, 1970), anion exchanger treatment (Japanese Patent Application No. 187896/1993), gel filtration method, dialysis and combinations of these methods.
The purification method of the present invention can be applied to any stage of these known purification methods. In particular, it is preferable to perform the cationic surfactant treatment of the present invention before the purification step performed by a column method such as lysine-affinity chromatography, ion exchanger treatment, gel filtration or the like, because the column can be prevented from clogging.

In the method of the present invention, it is preferable to remove the cationic surfactant from the plasminogen-containing liquid after the treatment with the cationic surfactant. The method of removal is not particularly limited, but the plasminogen and the surfactant can be separated by performing a chromatographic treatment under conditions under which plasminogen is adsorbed and surfactant is not adsorbed, such as lysine-affinity chromatography. can do. Further, various purification operations such as ion exchanger treatment and gel filtration can be performed between the cationic surfactant treatment and the lysine-affinity chromatography.

The plasminogen thus purified is used as a medicine. Therefore, the plasminogen is preferably subjected to a treatment for virus inactivation. Examples of the virus inactivating treatment include a method in which a liquid composition of plasminogen is heated at 50 to 100 ° C. for 5 to 30 hours, and a dry composition of plasminogen is 50 to 50 ° C.
A method of heating at 100 ° C. for 10 to 150 hours, a method of contacting with a trialkyl phosphate (for example, tri-N-butyl phosphate) (JP-A-60-51116, JP-A-3-218322), and these treatments. Combinations can be mentioned.

The purification method of the present invention makes it possible to obtain a plasminogen preparation in which impurities are effectively removed and which does not substantially exhibit an antihypertensive effect. In the present invention, "not substantially exhibiting a blood pressure lowering effect" means that the ratio of blood pressure after plasminogen administration to blood pressure before plasminogen administration is maintained at 85% or more.

The plasminogen preparation of the present invention is a solution,
It may be used as a suspension, an emulsion, or dissolved or suspended in a solvent at the time of use, and may contain a pharmacologically acceptable auxiliary component. Examples of auxiliary components include stabilizers, solubilizing agents, suspending agents, emulsifiers, buffers, preservatives,
An isotonicity agent and an excipient are exemplified.

The plasminogen preparation of the present invention is used for treating thrombosis and the like. The plasminogen preparation of the present invention can be administered to mammals including human,
It is parenterally administered by intravenous injection, subcutaneous injection, intracutaneous injection, intramuscular injection, bronchus, intraalveolar spray inhalation and the like. The dose of the plasminogen preparation of the present invention is
Although it varies depending on age, body weight, symptom, therapeutic effect, administration method, administration period, etc., 1 to 4000 cu / kg / day, preferably 10 to 400 cu / kg / day is exemplified.

[0024]

EXAMPLES The present invention will be described in detail below with reference to specific examples and test examples, but the present invention is not limited thereto.

Example 1 5 was added to the extraction residue of the Cohn II + III fraction of human plasma.
Double volume of extraction buffer [0.9 w / v% sodium chloride,
0.9 w / v% glycine buffer containing 10 KIE / ml aprotinin, or 0.1 M sodium chloride, 10 KI
E / ml aprotinin-containing 50 mM Tris buffer] was added, and the mixture was stirred at room temperature or under cooling for about 1 to 2 hours to extract lysyl-plasminogen. Aliquot the extract into a beaker and use cetylpyridinium chloride (CPC) or cetyltrimethylammonium bromide (CTAB) as the cationic surfactant, with final concentrations of 0.2, 0.4, 0.8w. / V%, and the mixture was stirred for about 0.5 to 1 hour. PH during extraction
Were 5.9, 6.9 and 8.5, respectively. Each extract was centrifuged at 10,000 g for 30 minutes, the supernatant was collected, and its appearance was visually observed to determine the clarity. Clarity was judged based on the following criteria. ◎ No turbidity is observed. ○ Slight turbidity is observed. × Remarkable turbidity is observed. The results of clarity determination are shown in Tables 1 and 2. The plasminogen activity recovery rate in the parentheses is [pH 5.0 during extraction, 100 plasminogen activity without surfactant addition.
% Activity recovery rate] is shown.

[0026]

[Table 1]

[0027]

[Table 2]

Example 2 In the same manner as in Example 1, human plasma was subjected to corn II + II.
Lysyl-plasminogen was extracted from the extraction residue of the I fraction. Aliquot the extract into a beaker and use cetylpyridinium chloride (CP) as a cationic surfactant.
C) was added so that the final concentrations thereof were 0.1 and 0.2 w / v%, respectively, and stirring was continued for about 0.5 to 1 hour. As a comparative example, polyoxyethylene sorbitan monooleate (Tween 80) was added instead of CPC so that the final concentrations thereof were 0.1 and 0.2 w / v%, respectively, and the same operation was performed. In both cases, the pH during extraction was set to 6.9. Centrifuge each extract for 30 minutes at 10,000 g,
The supernatant was collected and the clarity was determined in the same manner as in Example 1. Table 3 shows the results of the clarity determination. In parentheses is shown the plasminogen activity recovery rate (activity recovery rate when the plasminogen activity without addition of a surfactant is 100%).

[0029]

[Table 3]

Example 3 7.5 l of a 50 mM Tris buffer (pH 8.3) containing 0.9 w / v% sodium chloride was added to 1.5 kg of the extraction residue from the Cohn II + III fraction of human plasma or this fraction. Then, after stirring for 2 hours, cetylpyridinium chloride was added so that the final concentration was 0.2 w / v%, and stirring was further performed for 1 hour. The extract was centrifuged at 4000 rpm for 30 minutes to obtain a supernatant fraction of 5.5 liters. This supernatant was used as Toyo filter paper No. 2 (Toyo Roshi Kaisha, Ltd.), and the filtrate was applied to a lysine agarose (trade name: lysine Sepharose 4B) column equilibrated with 50 mM Tris buffer (pH 8.3) containing 0.9 w / v% sodium chloride. I applied.

After washing with 50 mM Tris buffer (pH 7.2) containing 1 M sodium chloride, elution was carried out with 50 mM Tris buffer (pH 7.2) containing 0.9 w / v% sodium chloride and 0.25 M lysine hydrochloric acid. 3.0 liters were obtained. Ultrafiltration membrane (trade name: minitan, molecular weight cutoff of 30,000,
Concentration and buffer exchange were carried out using 20 mM Tris buffer (pH 7.2) containing 0.45 w / v% sodium chloride and 0.2 w / v% lysine hydrochloride (Millipore).

Tri-N-butyl phosphate (TNBP) and Tween 80 were added to the concentrate at final concentrations of 0.3 w / v% and 1 w / v%, respectively, and 30
Warm at 6 ° C for 6 hours. This was used as a solution containing lysyl-plasminogen. QAE-Toyo previously equilibrated with 20 mM Tris buffer (pH 7.2) containing 0.45 w / v% sodium chloride and 0.2 w / v% lysine hydrochloric acid.
Ion exchange chromatography was performed on the obtained plasminogen using a pearl 550C column (φ113 mm × 10 cm: 1 liter, manufactured by Tosoh Corp.) [flow rate: 30 ml / min; equilibration and washing buffer: 0. 45w / v% sodium chloride, 0.2w / v%
20 mM Tris buffer (pH 7.2) containing lysine hydrochloric acid].

The unadsorbed fraction which passed through the column as it is,
It was applied to a lysine agarose (trade name: lysine sepharose 4B) column equilibrated with 20 mM Tris buffer (pH 7.2) containing 0.45 w / v% sodium chloride and 0.2 w / v% lysine hydrochloric acid. After washing with 50 mM Tris buffer (pH 7.2) containing 1 M sodium chloride,
It was eluted with 50 mM Tris buffer (pH 7.2) containing 0.9 w / v% sodium chloride and 0.25 M lysine hydrochloric acid, and the eluate was subjected to an ultrafiltration membrane (trade name: minitan, molecular weight cutoff of 30,000,
Millipore) and 0.45% sodium chloride, 0.
50 mM phosphate buffer with 2% lysine hydrochloric acid (pH 7.2)
Was used for concentration and buffer exchange. The resulting solution was sterilized and filtered to obtain a lysyl-plasminogen-containing solution.

Example 4 Lysyl-plasminogen obtained in Example 3 20 mg Sucrose 100 mg 0.45 w / v% sodium chloride, 0.2 w / v% lysine hydrochloric acid added 50 mM phosphate buffer (pH 7.2) 2 ml

The solution of and is dissolved in a membrane filter and then sterilized and filtered again. The filtered solution is aseptically dispensed into a vial and freeze-dried.
Heat treatment was performed at -80 ° C for 72 hours to prepare an intravenous injection.

Test Example 1 The effect of the lysyl-plasminogen obtained in Example 3 on the respiratory circulatory system was examined. Beagle dog (male, 6 months old,
Weight 8-10kg) Pentobarbital sodium 2
After anesthesia with 5 mg / kg (iv), the patient was fixed in the back position and a tracheal catheter was inserted to secure the airway. The respiratory rate and respiratory curve were measured by connecting to a coupler amplifier (AA-600H, Nihon Kohden) via a respiratory pickup (TR-612T, Nihon Kohden) attached to a tracheal catheter. A polyethylene catheter is inserted into the right femoral artery, and systolic and diastolic arterial pressures (SBP and DBP) are pressure transducers (Statham P-50, Gould).
Company). The electrocardiogram was measured by inputting lead II into the electrocardiograph. Heart rate (HR) is the electrocardiogram RR
The number of intervals was read from the recording paper. The above measurement items were input to a polygraph (RM-6000, Nihon Kohden), and changes during and after drug administration were continuously recorded on a recorder. The drug lysyl-plasminogen is 1000 cu
/ Kg was continuously administered at a dose rate of 8 ml / min for 5 minutes (40 ml / individual) from the right cephalic vein using a Harvard infusion pump. The circulatory dynamics were observed before drug administration and 5, 10, 15, 30, and 6 after the start of administration.
It was measured at 0 minutes. Lysyl-plasminogen administration group (4
Examples) include lysyl-plasminogen 1000 cu / k
g intravenously. To the control group (2 cases), 40 ml of physiological saline / individual is intravenously administered by the same method. In the statistical analysis, each measured value before and after drug administration is tested by the Dunnet method. The results are shown in Tables 4 and 5.

[0037]

[Table 4]

[0038]

[Table 5]

As shown in Tables 4 and 5, lysyl-plasminogen had no significant effect on any of the items of blood pressure, respiratory rate and heart rate. The plasminogen prepared through the purification step of the present invention has no side effects on the respiratory circulatory system such as a blood pressure lowering effect, and can be safely used as a medicine.

[0040]

According to the purification method of the present invention, a clear plasminogen-containing liquid can be obtained from a plasminogen-containing composition by adding a cationic surfactant. Therefore, even when purification is performed by the column method in the subsequent purification step, the column is prevented from being clogged and a large amount of raw material can be efficiently purified. According to the purification method of the present invention, the loss of plasminogen activity due to the addition of the cationic surfactant is small, and plasminogen can be recovered in high yield. Furthermore, the plasminogen composition prepared through the purification process of the present invention does not substantially exhibit side effects on the respiratory circulatory system such as a hypotensive action, and thus can be safely used as a pharmaceutical.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Tomohiko Matsui 2-25-1 Otani Otani, Hirakata, Osaka Prefecture Midori Central Research Institute Ltd. (72) Minoru Tsukada 2-25 Otani, Otani, Hirakata, Osaka No. 1 Stock Company Midori Cross Central Research Institute

Claims (8)

[Claims] 1. A method for purifying plasminogen, which comprises adding a cationic surfactant to a plasminogen-containing composition and collecting a clear solution. 2. The method according to claim 1, wherein the cationic surfactant is at least one selected from quaternary ammonium salts and alkylpyridinium salts. 3. The method according to claim 1, wherein the cationic surfactant is added so that the concentration of the cationic surfactant in the composition is 0.1 to 2.0 w / v%. 4. The method according to claim 1, wherein the plasminogen-containing composition has a pH of 5.5 to 9.0. 5. The method according to claim 1, wherein the cationic surfactant is removed after the cationic surfactant treatment. 6. The method according to claim 1, wherein the lysine-affinity chromatography treatment is performed after the cationic surfactant treatment. 7. A preparation containing plasminogen purified by a purification method, which comprises adding a cationic surfactant to a composition containing plasminogen and collecting a clear solution. 8. A plasminogen preparation having substantially no blood pressure lowering effect.