JPH0733799A - Thrombin having high specific activity and its production - Google Patents

Abstract

PURPOSE:To obtain the subject compound stable in a cold place for a long period and useful as a clinical medicine by converting a prothrombin composite material into to thrombin. CONSTITUTION:This thrombin can be obtained by allowing thromboplastin, snake poison, etc., to act on a prothrombin composite material (preferably human-deruved plasma protein-containing material, etc.) in a composite material state in the presence of about 0.0001 to 0.01M benzamidine (e.g. p- aminobenzamidine) and Ca<+2> and further treating it with a cation-exchanger (e.g. sufloethyl type such as sulfoethyl-dextran). The specific activity is >=1000 unit/A280.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to thrombin having a high specific activity and a method for producing the same. In particular, it relates to thrombin having a high specific activity and stability, and a method for producing the same.

[0002]

BACKGROUND OF THE INVENTION Thrombin is a serine protease having a molecular weight of about 34,000 and an isoelectric point of about 7.1, and is a proteolytic enzyme that acts at the final stage of the blood coagulation process. That is, it acts on fibrinogen to produce fibrin, which causes a blood coagulation action. Therefore, thrombin is clinically used as a local hemostatic agent in the surgical field and as a hemostatic agent for upper gastrointestinal bleeding in the medical field.

[0003] Thrombin exists in the form of prothrombin which is a precursor in the living body, and is produced by limited decomposition by activated factor X and the like. BACKGROUND ART Conventionally, thrombin has been produced by using human blood plasma as a raw material to first extract and purify prothrombin, and then allowing the obtained purified prothrombin to act on thromboplastin and the like. That is, the conversion to thrombin is performed on the purified prothrombin. As described above, the production of thrombin is carried out through various steps such as (1) purification of prothrombin, (2) conversion of prothrombin to thrombin, and (3) purification of thrombin as described above. is the current situation.

Various improvements have been reported based on this method. For example, a method in which citric acid-treated plasma is brought into contact with an anion exchanger, adsorbed prothrombin is converted to thrombin on the exchanger, and then eluted and recovered (US Pat. No. 5,138,389), plasma is treated with low temperature ethanol, A method in which prothrombin purified by treatment with an anion exchanger is converted into thrombin and further purified by treatment with a cation exchanger (JP-A-3-12).
8398) and the like are known.

However, the thrombin obtained by these methods has a problem that it has a low specific activity and is extremely unstable. Particularly, since stability is an essential condition for formulation, various methods have been conventionally taken to ensure the stability of thrombin.

[0006]

SUMMARY OF THE INVENTION An object of the present invention is to provide thrombin having a high specific activity. Another object of the present invention is to provide a method for producing thrombin having high specific activity and high stability.

[0007]

Means for Solving the Problems As a result of intensive studies to achieve the above object, the present inventors have found that a thrombin having a high specific activity can be easily obtained by using a prothrombin complex as a starting material for converting into thrombin. It was found that it can be manufactured. Furthermore, the present inventors have found that the thrombin obtained by such a method is stable in a cold state for a long time even in a solution state. The present inventors have completed the present invention by confirming that thrombin obtained by the method of the present invention has extremely high specific activity as compared with conventional thrombin.

That is, the present invention is 1000 units / A ₂₈₀
It is thrombin having a high specific activity as described above. Furthermore, the present invention is a method for producing thrombin, which comprises subjecting prothrombin to conversion into thrombin in a prothrombin complex state.

In the method of the present invention, the object to be treated for converting prothrombin into thrombin is the prothrombin complex. The prothrombin complex used herein contains prothrombin (blood coagulation factor II), which is one of the blood coagulation factors, and other factors related to blood coagulation. The components contained together with prothrombin include, for example, blood coagulation factor VII, factor IX,
Factor X and the like can be mentioned. Further, it does not have to be a high-purity refined product and may be a crude product.

The origin of the prothrombin complex and the method for obtaining the same are not particularly limited. In the method of the present invention, the prothrombin complex itself or all those containing the prothrombin complex can be used as the starting material for the conversion treatment. As the one containing a prothrombin complex, a plasma protein-containing material is preferably exemplified. In this case, for example, plasma itself, preferably plasma that has been appropriately purified to the extent that the complex is contained, can be used as a raw material for the conversion treatment. The origin of plasma is not particularly limited, and examples thereof include bovine origin and human origin, but human origin is preferable.

In the case of purifying plasma to obtain a prothrombin complex which is a raw material for the conversion treatment, known methods are used as a method for purifying plasma. For example,
JP-A-62-10019 and JP-A-3-128
The method described in Japanese Patent No. 398 can be used. In particular,
Examples thereof include a method of preparing a prothrombin complex by treating plasma with an anion exchanger, and a method of preparing a prothrombin complex using decryoplasma obtained by removing cryoprecipitate from plasma. As an anion exchanger, DE
Examples thereof include AE systems (eg DEAE-agarose, DEAE-dextran, DEAE-cellulose etc.), QAE systems (eg QAE-agarose, QAE-dextran etc.) and the like.

The prothrombin complex obtained by the above means can be subjected to a conversion treatment into thrombin in the state of the complex. As the conversion treatment, a known method for converting prothrombin into thrombin can be used.
For example, a method of causing thromboplastin, snake venom, etc. to act on prothrombin in the presence of Ca ⁺² , a natural conversion method using high-concentration citrate, and the like can be used.

In the present invention, it is particularly preferable that benzamidines (benzamidine, p-aminobenzamidine, etc.) are allowed to coexist during the conversion treatment. By allowing benzamidines to coexist, the conversion of prothrombin to thrombin can be carried out in a stable state, and thrombin can be obtained in good yield. The addition amount of benzamidines is, for example, about 0.0001 to 0.1M, preferably about 0.0001 to 0.01M.

The treated product obtained by the above conversion treatment is then subjected to a thrombin purification step. As such a purification method, any method can be used as long as it is a method capable of isolating thrombin converted in a complex state from the complex, for example, treatment with a cation exchanger, ammonium sulfate fractionation treatment, affinity Chromatographic treatment (for example, it is preferable to use arginyl methyl ester or heparin sodium as a ligand.)
The method etc. can be used. Preferably, it is treated with a cation exchanger (for example, Biochem., 10 (13), p25.
01, 1971, etc.). Examples of the cation exchanger used include sulfoethyl type (sulfoethyl-dextran, sulfoethyl-agarose etc.), sulfopropyl type (sulfopropyl-dextran, sulfopropyl-agarose etc.), carboxymethyl type (carboxymethyl-dextran, Carboxymethyl-agarose), Amberlite IRC50
Etc.

The cation exchanger treatment is carried out under the condition that thrombin is isolated as a purified product from other contaminants. Specifically, adsorption to a cation exchanger and washing are preferably carried out in a citrate buffer solution (pH: 0.01 to 0.05M) containing 0.01 to 0.1M sodium chloride.
6 to 8), or 0.01 to 0.2 M phosphate buffer (pH 6 to 8) containing 0.01 to 0.1 M sodium chloride. For elution, it is preferable to use a citrate buffer solution having a pH of about 6 to 8 at 0.05 to 0.3M.

Further purification can be carried out by known means such as dialysis, ultrafiltration and gel filtration.

In the present invention, a prothrombin complex-converted product is previously treated with a dialkyl phosphate or trialkyl phosphate having an alkyl group having 1 to 10 carbon atoms, particularly 2 to 10 carbon atoms, before the cation exchanger treatment. It is more preferable that the virus is inactivated (hereinafter referred to as SD treatment) by contact in the presence of a surfactant. SD processing is a known method (Japanese Patent Laid-Open No. 60-51116, Japanese Patent Laid-Open No. 3-218322).
No. publication). Further, particularly preferably, benzamidines (benzamidine, p-aminobenzamidine, etc.), basic amino acids (arginine, lysine, etc.) or ε-aminocaproic acid (EACA) are added as a stabilizer during SD treatment in a coexisting state. Is the way to do it. The amount of benzamidines added was 0.
0001-0.1M, 0.1-1 for basic amino acids
For example, about M, about 1 to 10% (w / v) in EACA (all are final concentrations).

The surfactant used during the SD treatment is preferably a nonionic surfactant such as a polyol fatty acid ester and a sorbitan fatty acid ester (Sp.
an), polyoxyethylene sorbitan fatty acid ester (Tween type), polyoxyethylene alkylphenol ether (Triton type), polyoxyethylene fatty acid ester, polyoxyethylene higher alkyl ether and the like. Specifically, Tween80
(Also known as polysorbate 80), Triton X100, sulfobetaine and the like are exemplified.

By the above treatment, various proteins that are contaminated (for example, fibrinogen, fibrin, α ₁ globulin,
Purified thrombin isolated from α ₂ globulin, β globulin, γ globulin, protease, etc.) can be obtained. The specific activity of such thrombin is at least 1.
It is preferably about 000 units / A ₂₈₀ . Here, 1 unit / A ₂₈₀ corresponds to 1 unit / mg protein. In addition,
The specific activity of thrombin can be determined by a usual measuring method. The method is preferably a method in which human plasma is used as a fibrinogen source and the coagulation time is measured by a BBL fibrometer or the like. Specifically, 0.1 ml of the sample diluted with physiological saline at various ratios was added to 3
It can be determined by keeping at 7 ° C. for 2 minutes, then adding 0.2 ml of plasma kept at 37 ° C., and measuring the coagulation time.

The thrombin can be made into a thrombin preparation having a high specific activity by formulating it by a method known per se. At this time, if necessary, addition of a pharmaceutically acceptable carrier, additives, etc. and heating, sterilization, sterilization filtration treatment, dispensing subdivision, freeze-drying treatment and the like are carried out according to ordinary methods.

[0021]

The thrombin prepared according to the present invention is
It has a high specific activity of at least 1000 units / A ₂₈₀ . Conventional and commercial thrombin preparations have a specific activity of 5
Since it is about 00 to 600 units / A ₂₈₀ , the thrombin of the present invention is extremely useful as a thrombin preparation. In addition, the thrombin prepared by the present invention is stable in a cold place for a long time even in a solution state. Therefore, the method of the present invention is extremely practical as a method for producing thrombin, and the prepared purified thrombin can be clinically provided as a useful drug.

[0022]

EXAMPLES Examples will be given to explain the present invention in more detail, but the present invention is not limited thereto.

Example 1 Preparation of Prothrombin and Thrombin 2 liters of decryogenic plasma obtained by freeze-thawing human plasma was equilibrated with a buffer solution (pH 7) consisting of 0.075 M sodium chloride and 0.01 M sodium citrate. After contacting with DEAE-dextran (trade name: DEAE-Sephadex, A-50, manufactured by Pharmacia) to adsorb the prothrombin complex, a buffer solution (pH 7) consisting of 1M sodium chloride and 0.01M sodium citrate. Eluted at. After dialysis of the eluate, thromboplastin, 2.5% calcium chloride and 0.082M
Benzamidine was added in an amount of 1/10 each and heated at 25 ° C. for 3 hours. Adjusted pH to 7, tri-N-butyl phosphate (TNBP), Tween 80 and EA
The final concentrations of CA were 0.3% (w / v) and 1% (w, respectively).
/ V) and 4% (w / v), and add 30
Warm at 6 ° C for 6 hours. Next, the reaction solution was adjusted to pH 6.7.
Adjusted to 0.02M sodium citrate (pH 6.
Thrombin alone was adsorbed by contacting it with sulfopropyl-dextran (trade name SP-Sephadex C-50, manufactured by Pharmacia) equilibrated in 7). The column was loaded with 0.02M sodium citrate solution (pH
6.7), washed with 0.1 M phosphate buffer (pH 6.5) and then with 0.045 M sodium citrate (pH 7) solution, and then adsorbed thrombin with 0.1 M sodium citrate solution (pH 7). Eluted and isolated. The eluate was concentrated with Pellicon (molecular weight cut-off 10,000: manufactured by Millipore) and sterilized by filtration to prepare purified thrombin. Table 1 shows the degree of purification (specific activity, recovery rate) of prothrombin and thrombin obtained in each purification step.

[0024]

[Table 1]

Example 2 Preparation of prothrombin and thrombin Decryoplasm obtained by freeze-thawing human plasma was 0.07
Contact with DEAE-Sephadex A-50 equilibrated with a buffer consisting of 5M sodium chloride and 0.01M sodium citrate (pH 7) to form a buffer consisting of 0.15M sodium chloride and 0.01M sodium citrate (pH 7). ), A buffer consisting of 1M sodium chloride and 0.01M sodium citrate (pH
It was eluted in 7). Add 1/5 each of 2.5% calcium chloride and thromboplastin to the eluate
It was heated at 5 ° C for 3 hours. The pH was adjusted to 7 and Tri-N-
Butyl phosphate (TNBP), Tween 80 and EACA were each 0.3% (w / v) final concentration, 1
% (W / v) and 4% (w / v) were added, and the mixture was heated at 30 ° C. for 6 hours. Next, the reaction solution is adjusted to pH.
Was adjusted to 6.7 and 0.02M sodium citrate (p
H6.7) was contacted with sulfopropyl-dextran (trade name SP-Sephadex C-50, manufactured by Pharmacia) to adsorb only thrombin.
The column was loaded with 0.02 M sodium citrate solution (p
H6.7), 0.1 M phosphate buffer (pH 6.5),
Then, after washing with a 0.045 M sodium citrate (pH 7) solution, the adsorbed thrombin was eluted and isolated with a 0.1 M sodium citrate solution (pH 7). The eluate was concentrated with Pellicon (molecular weight cutoff 10,000: manufactured by Millipore),
Sterilization filtration was performed to prepare purified thrombin.

Comparative Example 1 Preparation of thrombin from putrombin alone Prothrombin was extracted from the corn No. II + III extraction fraction with 0.1 M sodium chloride solution in the form of a simple substance. After adjusting the pH of the obtained extract to 6.7, 1% calcium chloride (1/10 amount) and thromboplastin (1/10 amount)
And plasma (1/40 volume) was added and heated at 20 ° C. for 3 hours. This reaction solution was added with 0.1 M phosphate buffer (pH 6.
5) contacting with the anion exchanger (SP-Sephadex C-50, Pharmacia) equilibrated in 5),
The adsorbed thrombin was eluted with 0.1 M citrate buffer (pH 6.7). In the eluate, 0.3% (w /
v) TNBP, 1% (w / v) Tween 80 and 4
% (W / v) EACA was added, and the mixture was heated at 30 ° C. for 6 hours (SD treatment). The obtained reaction solution was applied to an affinity column (column: Heparin Toyopearl, manufactured by Tosoh Corporation) equilibrated with 0.02M citrate buffer (pH 7.0), and 0.3M citrate buffer ( pH 6.0)
The thrombin was eluted at. This eluate was concentrated with an ultrafiltration membrane (molecular weight cutoff of 10,000) and sterilized by filtration to prepare purified thrombin.

Experimental Example 1 Each property of the thrombin of the present invention obtained in Example 1 was
The comparison was made with a thrombin purified product prepared from the corn II + III extraction fraction obtained in Comparative Example 1 and a commercially available thrombin preparation (manufactured by KEIKAKEN). (1) Specific activity The thrombin titer and the absorbance at 280 nm were measured,
The specific activities were compared. As a result, the thrombin of the present invention is 1000 units / A ₂₈₀ , whereas the thrombin of the present invention is II + III.
555 units / A ₂₈₀ derived from the extracted fraction, 5 commercially available
82 was units / A _{28 0.} (2) Distribution As a result of cellulose acetate electrophoresis, the thrombin of the present invention has 100% β fraction, whereas
The extraction fraction was 55%, and the commercial product was 92%. (3) Molecular weight measurement As a result of SDS-PAGE electrophoresis (Fast System, manufactured by Pharmacia), the thrombin of the present invention was confirmed as a single band with a molecular weight of 34,000 under non-reducing conditions.
In addition to the band with a molecular weight of 34,000, bands derived from the II + III extracted fraction and the commercial product were observed on the high molecular side and the low molecular side. Gel filtration analysis by high performance liquid chromatography (column: TSKgel G3000SWXL (manufactured by Tosoh Corporation), mobile phase: 0.2M NaCl-containing 0.1M phosphate buffer (pH 6.8), flow rate: 0.8 ml / min, detection wavelength: 280 nm) As a result, the thrombin of the present invention was obtained as a single peak at a retention time of around 13.2 minutes, whereas the thrombin of the present invention derived from the II + III extracted fraction and a peak at a retention time of around 13.2 minutes were obtained. Besides, a contaminated peak appeared. (4) Stability 500 units / ml of thrombin of Example 1 and 170
When stored in a solution of 0 unit / ml at 25 ° C. for 7 days, the thrombin titer was stable without any decrease. On the other hand, when a commercially available product was similarly stored in a solution state of 1000 units / ml, the titer decreased by about 20%.

[0028]

[Table 2]

Experimental Example 2 Stability of thrombin of Example 2 Using the thrombin obtained in Example 2, sample solutions of 6000 units / ml, pH 6, 1000 units / ml, pH 5.5 and pH 6 were prepared, 8 at 10 ° C and 25 ° C
The remaining rate of thrombin titer after storage for a week was examined. As a result, the thrombin prepared by the method of the present invention was at least 8% in solution and in ordinary refrigerated storage (10 ° C).
The week turned out to be stable.

[0030]

[Table 3]

Reference Example 1 Examination of Conditions for Conversion Reaction to Thrombin Various stabilizers were added 1 hour after the start of the conversion reaction to thrombin, and from the residual rate of thrombin activity 4 hours after the addition, the stability of the stabilizer was determined. The stabilizing effect on thrombin was investigated

[0032]

[Table 4]

A conversion reaction to thrombin (25 ° C., 3 hours) was carried out with the stabilizer added from the beginning. Table 4
Shows the stabilizing effect of various stabilizers on thrombin.

[0034]

[Table 5]

As a result of the above and above, the stabilizing effect when the concentration of benzamidine having the most effect was changed was examined.

[0036]

[Table 6]

Reference Example 2 Stabilizing conditions during SD treatment were examined in the presence of various additives. The additives used and their concentrations are listed in Table 7. It was treated at 30 ° C. for 6 hours in the presence of these additives. The results are shown in Table 7.

[0038]

[Table 7]

The relationship between the stabilizing effect of arginine (Table 8) and EACA (Table 9) as stabilizers and the stabilizing effect was confirmed.

[0040]

[Table 8]

[0041]

[Table 9]

When arginine is added, the final concentration is 0.
A stabilizing effect was observed at 4 M or more. Further, in the case of EACA, it was found that the addition of 1% w / v or more has a stabilizing effect.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Yasuaki Morisada 3-5-44, Miyajima Shimadori, Miyakojima-ku, Osaka City Midori Cross Miyakojima Plant (72) Inventor Yuuhiko Matsumoto Nakajima, Miyajima, Miyakojima-ku, Osaka 3-5-44 Midori Cross Miyakojima Plant Stock Company

Claims (3)

[Claims] 1. Thrombin having a specific activity of 1000 units / A ₂₈₀ or more. 2. A method for producing thrombin, which comprises subjecting a prothrombin complex to conversion treatment into thrombin. 3. A method for producing thrombin, which comprises recovering thrombin by subjecting a prothrombin complex to conversion treatment into thrombin, and treating the obtained treated product with a cation exchanger.