JPH11228443A - Novel method for activating prothrombin and method for producing thrombin based on the method - Google Patents

Abstract

(57) [Summary] [Object] In a reaction for activating prothrombin to thrombin, a raw material which is easily available as a "prothrombin complex" or "aqueous solution containing prothrombin and blood coagulation factor X as main components" as a prothrombin-containing aqueous solution. The present invention provides a method for efficiently producing thrombin on an industrial scale based on means for achieving conversion to thrombin even in the absence of thromboplastin. [Constitution] Glycerin,
Alternatively, the treatment is performed with glycerin in the presence of a calcium salt.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing thrombin. More specifically, when thrombin is obtained on an industrial scale, a method capable of activating prothrombin to thrombin by performing a treatment with glycerin or glycerin and a calcium salt even in the absence of thromboplastin, and the activation step And a method for producing thrombin.

[0002]

BACKGROUND OF THE INVENTION Thrombin is a serine protease with a molecular weight of 37,000 and is a proteolytic enzyme that functions in the final stage of the blood coagulation reaction. Thrombin coagulates blood by acting on fibrinogen to produce fibrin.
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. Further, it is also applied as one component of a fibrin adhesive.

[0003] Thrombin is present in the circulating blood in the form of its precursor prothrombin, and activated X
Generated by subjecting to limited decomposition by factors. Conventionally, thrombin is produced using human and bovine plasma as raw materials,
First, a method has been performed in which prothrombin is extracted and purified, and thrombin is allowed to act on the obtained purified prothrombin to obtain thrombin. That is, conventionally, conversion to thrombin has been performed on purified prothrombin. The obtained thrombin is further purified through various steps. Further, as an improved method based on this method, "plasma treated with low-temperature ethanol and then anion exchanger, and the resulting prothrombin is converted to thrombin,
Further, a method of purifying by cation exchanger treatment (JP-A-3-128398) is known.

[0004] Thromboplastin, which is mainly prepared from human placenta, is used, but it is difficult to procure the raw materials and it cannot be said that a sufficient amount is always available. In addition, it cannot be completely removed in the manufacturing process, and may be mixed into the final product. As a method for converting prothrombin to thrombin, in addition to the above method using thromboplastin, a method using snake venom, a method using high-concentration citrate (Japanese Patent Application Laid-Open Nos. 4-365481 and 5-194261). Etc. are known. However, when snake venom is used, similarly to thromboplastin, it is difficult to procure raw materials, and at the same time, there is a concern that contamination with the final product may occur. In addition, the spontaneous conversion method using high-concentration citrate has an advantage that contamination of tissue proteins such as thromboplastin is avoided, but it cannot be said that sufficient conversion efficiency is guaranteed for an industrial production method.

[0005] JP-A-5-186369, JP-A-6-186369
46852 and International Patent Publication WO 95/3153
No. 6 discloses a method for converting prothrombin to thrombin with calcium chloride. Japanese Patent Application Laid-Open No. 7-33799 discloses a method in which a prothrombin to be converted into thrombin is not a purified prothrombin but a prothrombin complex. Although these methods can be effective production methods under limited conditions, they cannot be said to be simple and effective methods.

Under such circumstances, an object of the present invention is to establish an efficient method for producing thrombin on an industrial scale. That is, an object of the present invention is to provide a method for easily obtaining raw materials and efficiently converting prothrombin to thrombin when obtaining thrombin on an industrial scale.

[0007]

Means for Solving the Problems, Structure of the Invention The inventors of the present invention have made intensive studies in view of the above-mentioned problems, and as a result, have completed the present invention which provides an efficient method for producing thrombin. Hereinafter, the present invention will be described in more detail.

The present invention relates to an activation method capable of achieving the conversion of prothrombin to thrombin even in the absence of thromboplastin, and a method for producing thrombin including the activation step. As a preferred embodiment of the method of the present invention, the object subjected to the conversion process to thrombin,
"Prothrombin complex" or "aqueous solution containing prothrombin and blood coagulation factor X as main components". The prothrombin complex used here contains prothrombin (blood coagulation factor II), which is one of the blood coagulation factors, and other factors related to blood coagulation. Components included with prothrombin include blood coagulation factor VII, factor IX, and factor X. The object to be subjected to the above-mentioned conversion into thrombin need not be a highly purified product, but may be a crude product. "Prothrombin complex" or "Aqueous solution containing prothrombin and blood coagulation factor X as main components"
Examples of the preparation method 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. . There is no particular limitation on the origin of the plasma used as a raw material, and examples thereof include those derived from cattle and those derived from humans, and are preferably derived from humans.

[0009] The "prothrombin complex" or "aqueous solution containing prothrombin and blood coagulation factor X as the main components" obtained as described above is treated at 4 to 37 ° C, preferably 10 to 37 ° C.
An activation reaction from prothrombin to thrombin is performed by treatment with glycerin or glycerin and a calcium salt at 25 ° C. The prothrombin concentration in the reaction solution is, for example, about 20 to 120 units / ml. Here, the unit / ml means the amount of prothrombin contained in 1 ml of plasma. The treatment with glycerin or glycerin and a calcium salt referred to in the present invention refers to an activation reaction to thrombin by adding and mixing glycerin or glycerin and a calcium salt to an aqueous solution mainly containing prothrombin and blood coagulation factor X. Is what you do. Specifically, a preferred embodiment is a method in which a glycerin solution or a glycerin solution and a calcium salt solution are added to an aqueous solution mainly containing prothrombin and blood coagulation factor X, and the mixture is stirred, mixed, and left. Although the calcium salt can be added as a solid, it is preferable to add a solution.

Glycerin has a molecular weight of about 92.1, a high viscosity, a high moisturizing property, and is chemically stable. Glycerin used in the present invention is a high-purity product that can be added to pharmaceutical production, and the addition of glycerin stabilizes thrombin formed in the activation reaction and suppresses inactivation of thrombin due to autolysis. This contributes to efficient activation to thrombin. The processing concentration of glycerin is preferably about 1 to 30%. Examples of the calcium salt include calcium chloride, calcium hydroxide, calcium acetate and the like. The treatment concentration of the calcium salt is 0 to 100 mM, and preferably about 5 to 25 mM. The treatment time is, for example, about 1 to 20 days, but it is appropriately increased or decreased depending on the composition of the reaction solution.

A method for producing thrombin, which comprises converting prothrombin to thrombin by treating with a calcium salt, is described in Japanese Patent Application Laid-Open No. 5-186369,
JP-A-6-46852 and International Patent Publication WO95 /
No. 31,536. However, there was no finding suggesting that glycerin treatment in the presence of a calcium salt efficiently promotes the activation reaction from prothrombin to thrombin. Therefore, it can be said that the present invention provides novel knowledge different from the above-described prior art in both aspects of the method and the effect. Further, according to the invention of the present application, it is possible to achieve the object of "easy and efficient conversion of prothrombin to thrombin when raw materials are obtained on an industrial scale".

[0012] Thrombin obtained by the activation reaction is purified by a known method. As a purification method, a cation exchanger treatment is exemplified, but further purification can be performed by other known methods. The thrombin is formulated by a method widely known to those skilled in the art. At this time, if necessary, a pharmaceutically acceptable carrier, an additive or the like is added and sterilization filtration, dispensing, freeze-drying, heating, etc. are performed according to a usual method.

[0013]

According to the present invention, thrombin can be efficiently prepared on an industrial scale using easily available raw materials. Hereinafter, the present invention will be specifically described with reference to test examples and examples, but the present invention is not limited to these examples.

[0014]

EXAMPLES Preparation Example 1 (Preparation of Prothrombin) Cryo supernatant obtained from human plasma was adsorbed on an anion exchange resin. The fraction containing prothrombin and factor X is eluted and collected with a buffer (pH 7.0) containing 0.7 M sodium chloride, and a final concentration of 10% polyethylene glycol 4,000 (to remove unnecessary substances) of 4,000 ( Prothrombin and coagulation factor X were roughly purified by PEG fractionation by addition of PEG 4,000). Furthermore, the purity of prothrombin obtained by purification by Q-Sepharose FF chromatography is about 6.
0 plasma units / A280.

[0015] obtained in Example 1 Preparation Example 1 Prothrombin about 50 plasma units / ml
(Containing about 148 plasma units / ml of factor X)
0% (w / v) glycerin and 5 mM calcium chloride were added, and reacted at 25 ° C. and pH 7.5. The activity of the purified thrombin was measured as the clotting activity using fibrinogen at each treatment time. The unit of thrombin activity used thereafter is the American standard (U.
S. Standard Thrombin (formerly NIH standard product). The results are as shown in FIG. 1, and activation to thrombin was achieved in 7 to 10 days.

Test Example 1 About 50 plasma units / m of prothrombin obtained in Preparation Example 1
Glycerin of various predetermined concentrations is added to the activation reaction solution containing 1 and 5 mM calcium chloride at 25 ° C., pH 7.
The reaction was carried out at 5. Activation to thrombin in the presence of various concentrations of glycerin is as shown in FIG. When the glycerin treatment concentration was 5% (w / v) or more, the effect of promoting the activation reaction was observed as compared with the case where glycerin was not added. Especially at 10 to 25% (w / v), effective activation to thrombin was achieved.

Test Example 2 The relationship between the concentration of calcium chloride in the activation reaction and the activation to thrombin was examined. The concentration of glycerin to be added was selected to be 20% (w / v). About 50 plasma units / ml of prothrombin obtained in Preparation Example 1 and various concentrations of calcium chloride were mixed and reacted at 25 ° C. and pH 7.5. Activation of thrombin at each concentration of calcium chloride is shown in FIG.
As shown in FIG. The optimal concentration of calcium chloride is 5
５０50 mM.

Test Example 3 The effect of prothrombin concentration on activation was examined. The concentration of glycerin to be added was 20% (w / v), the concentration of calcium chloride was 5 mM, and the prothrombin obtained in Preparation Example 1 was adjusted to various predetermined concentrations.
The activation reaction was performed at pH 7.5. Activation to thrombin at various concentrations is as shown in FIG. FIG. 5 shows the activation rate. Here, the activation rate refers to the thrombin activity (NI) obtained per plasma unit of prothrombin (prothrombin activity contained in 1 ml of normal human plasma).
H unit). Optimal concentration of prothrombin is 25-1
00 plasma units / ml.

Test Example 4 The effect of temperature on the activation reaction was examined. The final concentration of glycerin to be added was 20% (w / v), the concentration of calcium chloride was 5 mM, and the prothrombin obtained in Preparation Example 1 was adjusted to about 50 plasma units / ml.
The activation reaction was performed with H7.5. Activation to thrombin at each temperature is as shown in FIG. The optimum temperature for the activation reaction was found to be 10 to 25 ° C.

[0020]

[Brief description of the drawings]

FIG. 1 is a diagram showing an activation reaction by adding glycerin in the presence of calcium.

FIG. 2 is a graph showing the effect of glycerin concentration on the activation reaction in the presence of 5 mM calcium chloride.

FIG. 3 is a diagram showing the influence of the concentration of calcium chloride on the activation reaction.

FIG. 4 is a diagram showing the influence of the concentration of prothrombin on the activation reaction.

FIG. 5 is a graph showing the relationship between prothrombin concentration and activation rate.

FIG. 6 is a diagram showing the effect of temperature on the activation reaction.

Claims (12)

[Claims] 1. A method for activating prothrombin to thrombin, comprising treating an aqueous solution containing prothrombin with glycerin. 2. The method for activating prothrombin to thrombin according to claim 1, wherein the aqueous solution containing prothrombin is treated with glycerin in the presence of a calcium salt. 3. The method for activating prothrombin to thrombin according to claim 1 or 2, wherein the treatment with glycerin or glycerin and a calcium salt is performed in the absence of thromboplastin. 4. The method for activating prothrombin to thrombin according to claim 1, wherein the treatment temperature with glycerin or glycerin and a calcium salt is 4 ° C. to 37 ° C. 5. The method for activating prothrombin to thrombin according to claim 4, wherein the treatment temperature with glycerin or glycerin and a calcium salt is 10 to 25 ° C. 6. The concentration of glycerin is in a final concentration of 1 to 30%.
The method for activating prothrombin to thrombin according to any one of claims 1 to 5, which is (w / v). 7. The method for activating prothrombin to thrombin according to claim 2, wherein the coexisting calcium salt is selected from calcium chloride, calcium hydroxide and calcium acetate. 8. The concentration of the calcium salt is adjusted to a final concentration of 0 to 100.
8. The method for activating prothrombin to thrombin according to claim 2, wherein the amount is mM. 9. The method for activating prothrombin to thrombin according to any one of claims 1 to 8, wherein the prothrombin-containing composition is a prothrombin complex. The method for activating prothrombin to thrombin according to any one of claims 1 to 8, wherein the prothrombin-containing composition is an aqueous solution containing prothrombin and blood coagulation factor X as main components. A method for producing thrombin, comprising a step of activating prothrombin to thrombin according to any one of claims 1 to 10. 12. The method for producing thrombin according to claim 11, wherein a cation exchanger treatment is performed after the step of activating prothrombin to thrombin according to any one of claims 1 to 10.