JPH10150980A - Purification of prothrombin - Google Patents

Abstract

PROBLEM TO BE SOLVED: To conduct a fine purification of the subject compound for local hemostatic or the like by developing a solution containing prothrombin through an anion exchange-resin for adsorbing the prothrombin thereto and subsequently eluting it with a buffer solution containing a calcium salt. SOLUTION: This high-purity prothrombin useful as a local hemostatic in the surgical field, hemostatic for upper alimentary canal, etc., in the medical field and a constituent of tissue adhesive is obtained by developing a solution containing prothrombin (occasionally referred to as thromboplastic factor II) through an anion exchange-resin so as to adsorb the prothrombin thereto, followed by eluting it with a buffer solution containing a calcium salt; or by controlling the ionic strength of the solution containing the prothrombin so as to allow the prothrombin to be adsorbed to the anion exchange-resin in the absence of calcium salts and subsequently preparing the solution to contain the calcium salt to develop it through the anion exchange-resin so that the prothrombin freely passes therethrough.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for purifying prothrombin (sometimes referred to as blood coagulation factor II). More specifically, when obtaining prothrombin on an industrial scale, or when further obtaining thrombin from the prothrombin, prothrombin is inhibited by inhibiting the adsorption of prothrombin to an anion exchange resin by a calcium salt. It relates to a method of purification.

[0002]

2. Description of the Related Art Prothrombin is a serine protease precursor having a molecular weight of about 72,000, and is activated by activated factor X in the blood coagulation reaction to be converted to thrombin. Thrombin is a serine protease having a molecular weight of about 37,000 and is a proteolytic enzyme that works 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. It is also applied as a component of a tissue adhesive (fibrin adhesive).

[0003] In the production of thrombin, first, prothrombin is extracted from human or bovine plasma as a raw material.
Purified. Thereafter, the prothrombin fraction can be converted to thrombin by a suitable activation method. The prothrombin fraction used at this time contains prothrombin represented by a prothrombin complex as a main component and contains blood coagulation factor X and the like that contribute to the activation reaction.
It has been known that the specific activity of thrombin purified through various steps after the activation reaction depends on the purity of prothrombin subjected to the activation reaction. That is, it has been revealed that it is advantageous to convert prothrombin into thrombin with high purity.

A method for purifying prothrombin based on the prior art is disclosed in Japanese Patent Application Laid-Open (JP-A-3-128398).
No.). This method is, specifically,
In this method, decryoplasma obtained by removing cryoprecipitate from plasma is treated with an anion exchanger, and prothrombin is prepared by elution of sodium chloride. However, the purity of the prothrombin in the prothrombin fraction thus obtained is not satisfactory. In addition, in the prothrombin obtained by the conventional anion exchanger treatment, there is a substance showing an inhibitory activity of serine protease as an impurity protein, which inhibits the reaction upon activation of prothrombin to thrombin. In addition, it inhibits the generated thrombin and causes a decrease in the thrombin yield. Although it is possible to purify prothrombin as a research material with high purity, those methods cannot be applied to the purification of prothrombin on an industrial scale.

[0005] Under such circumstances, an object of the present invention is to establish an efficient method for purifying prothrombin on an industrial scale. That is, an object of the present invention is to provide a method for easily and efficiently chromatographically purifying prothrombin when obtaining prothrombin on an industrial scale or when obtaining thrombin having higher specific activity from the prothrombin. .

[0006]

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 relating to an efficient method for purifying prothrombin and a method for producing prothrombin based on the purifying method. . Less than,
The present invention will be described in more detail. The gist of the present invention is:
A method for purifying prothrombin, which utilizes a phenomenon in which the adsorption of prothrombin to an anion exchange resin is strongly inhibited by a calcium salt. The method for purifying prothrombin of the present invention is characterized in that the calcium ion binding property of prothrombin is based on. That is, a solution containing prothrombin is developed on an anion exchange resin to adsorb prothrombin, and prothrombin is eluted with a buffer containing a calcium salt. Alternatively, the ionic strength of the solution containing prothrombin is set to an ionic strength at which prothrombin can be adsorbed to an anion exchange resin in the absence of a calcium salt, for example, a concentration of 0.2 M or less in terms of sodium chloride concentration. And then developed on the anion exchange resin to allow prothrombin to pass through to obtain a desired prothrombin. Furthermore, the present inventors have found that prothrombin obtained by such a method has no serine protease inhibitory activity typified by trypsin inhibitory activity, is extremely advantageous as a raw material to be converted to thrombin, and as a result, It has been found that thrombin having a high specific activity can be easily produced.

[0007] In the method of the present invention, the target substance as a starting material of prothrombin is "plasma" or "decryoplasma obtained by removing cryoprecipitate from plasma".
The origin of the plasma is not particularly limited, and examples thereof include those derived from cattle and those derived from human, and preferably those derived from human. These targets can be directly or anion-exchanger-adsorbed in the step of recovering a prothrombin complex, PEG fractionation described in Japanese Patent Application Publication No. 45-50358, and other crude fractionation methods. After undergoing any one of the above methods or a method combining them, it is subjected to the anion exchanger treatment of the present invention. The prothrombin complex used herein contains prothrombin and other factors related to blood coagulation. Components included together with prothrombin include blood coagulation factor VII, factor IX, factor X, and the like.

[0008] The anion exchange resin used in the present invention is not particularly limited, but is preferably DEAE Sepharose Fast Flow (DEAE) manufactured by Pharmacia.
-Sepharose FF) and Q Sepharose Fast Flow (Q-Sepharose FF) are used. Examples of the calcium salt that inhibits the adsorption of prothrombin on the anion exchange resin include calcium chloride, calcium hydroxide, calcium acetate and the like. 100 mM calcium salt concentration
Although it is as follows, about 1 to 20 mM is preferable in order to enhance the ability to separate prothrombin and a substance having trypsin inhibitory activity. High-purity prothrombin obtained by the purification method of the present invention is subjected to a conversion treatment into thrombin.
As the conversion treatment, a “method of causing thromboplastin to act in the presence of a calcium salt” (Thromb. Haemost., V
ol.42, p.972-982, 1979), "How to make snake venom work"
(J. Biochem. (Tokyo), vol.83, p.559-570, 1978),
In addition to known methods such as "natural conversion method using high-concentration citrate" (JP-A-4-365481, JP-A-5-194261), the inventors of the present invention have clarified prior to the present invention. The "method of causing PEG to act in the presence of a calcium salt" (Japanese Patent Application No. 8-227695) can be used, but the "method of causing PEG to act in the presence of a calcium salt" is particularly preferred. The processed product obtained by the conversion to thrombin is then subjected to a thrombin purification step. As such a purification method, a cation exchanger treatment is exemplified, but it can be further purified by other known methods.

The specific activity of thrombin obtained through the purification step is preferably 1,000 NIH units / A280 or more. Here, the NIH unit corresponds to the unit of thrombin specified in the Japanese Pharmacopoeia, and A280 means the absorbance of the protein solution at a wavelength of 280 nm. The thrombin is
A thrombin preparation having a high specific activity can be obtained by a method known per se. At this time, if necessary, addition of a pharmaceutically acceptable carrier, additives, and the like, sterilization filtration, dispensing, freeze-drying, heating, and the like are performed according to ordinary methods.

[0010]

Industrial Applicability According to the present invention, prothrombin is purified on an industrial scale with high purity using a general-purpose gel carrier called anion exchange resin, and serine protease inhibitory activity that inhibits the activation reaction to thrombin is removed from prothrombin. It is also possible to do. Furthermore, prothrombin obtained by the purification method of the present invention is efficiently activated to thrombin. According to the present invention, it is possible to achieve the purpose of `` purifying prothrombin from a prothrombin-containing solution as a raw material to high purity without requiring special materials when obtaining prothrombin and / or thrombin on an industrial scale ''. it can. Hereinafter, the present invention will be described specifically with reference to Examples and Comparative Examples, but the present invention is not limited to these Examples.

[0011]

EXAMPLES Prior to the description of the examples, methods for measuring prothrombin activity, trypsin inhibitory activity and thrombin activity used in the present invention will be outlined. 1) Prothrombin activity Prothrombin activity was determined by activating prothrombin with ecarin (snake venom) and converting it to thrombin, and then measuring the amount of decomposition of a specific synthetic substrate S-2238 (first chemical) for thrombin (blood). And vascular, first
Vol. 2, No. 1, pp. 144-151, 1981). A calibration curve was prepared from normal human plasma (1 ml of normal human plasma contained 1 plasma unit of prothrombin) treated in the same manner as the specimen, and the prothrombin activity value was expressed as "plasma units / ml".

2) Trypsin inhibitory activity Trypsin inhibitory activity is described in a reference (Methods in Enzym).
ology, vol. 80, p. 754-765, 1981). Trypsin is a chromogenic substrate (Nα-Benzoyl-DL
-Arginine p-Nitroanilide: BAPNA) decomposes to 40
The increase in absorbance at 5 nm is measured. Aprotinin having trypsin inhibitory activity reduces this change in absorbance in a concentration-dependent manner. In a certain aprotinin concentration range,
“Aprotinin concentration” and “A minute change in absorbance (ΔA40
5 / min) "indicates a linear relationship. Using this as a calibration curve, the trypsin inhibitory activity of the sample was examined, and the trypsin inhibitory activity in the sample was represented by "KIE / ml" as a value corresponding to aprotinin activity.

3) Thrombin activity The thrombin activity was measured according to the fibrinogen clotting time measurement method described in the Japanese Pharmacopoeia (NIH unit).
Further, the thrombin activity obtained per prothrombin plasma unit was expressed as "NIH unit / prothrombin 1 plasma unit" as an activation rate.

Example 1 Decryoplasma obtained from human plasma was adsorbed on an anion exchange resin. The prothrombin complex is eluted and recovered with a buffer (pH 7.0) containing 0.7 M sodium chloride,
10% polyethylene glycol 4,000 (PEG 4,0
The prothrombin complex was roughly purified by PEG fractionation to remove the insoluble material in (00).

Comparative Example 1 The prothrombin complex obtained in Example 1 was
It was dialyzed against a Na ₃ citrate (pH 6.0) buffer, adsorbed on a Q-Sepharose FF column equilibrated with the same buffer, and washed to remove unbound proteins. The fraction containing prothrombin was purified with 25 mM Na ₃ citrate (p
Elution was performed by linearly loading a 0-1.0 M NaCl gradient in H6.0) buffer. The elution pattern of total protein, the elution pattern of prothrombin, and the NaCl concentration gradient represented by the A280 value are as shown in FIG. Prothrombin was eluted as one main peak together with contaminating proteins having high adsorption characteristics to the anion exchange resin, and the purification efficiency by the chromatography step was not high.

Comparative Example 2 The prothrombin complex obtained in Example 1 was
Q-Sepharose dialyzed against a buffer of Na ₃ citrate 0.2M NaCl (pH 6.0) and equilibrated with the same buffer
After adsorbing to the FF column, the column was washed to remove unbound proteins. 20 mM Tris-HCl
After replacing with 0.2 M NaCl (pH 7.5), the fraction containing prothrombin was subjected to 20 mM Tris-HCl /
Elution was carried out with a buffer of 0.5 M NaCl (pH 7.5). Prothrombin recovery in column chromatography,
The purity (specific activity) of the obtained prothrombin and the trypsin inhibitory activity value present in the obtained prothrombin fraction are as shown in Table 1.

[0017]

[Table 1]

Example 2 The prothrombin complex obtained in Example 1 was added to 25 mM
Q-Sepharose dialyzed against Na ₃ citrate / 0.2 M NaCl (pH 6.0) buffer and equilibrated with the same buffer
After adsorbing to the FF column, the column was washed to remove unbound proteins. 20 mM Tris-HCl
After replacing with a buffer solution of 0.2 M NaCl (pH 7.5), the fraction containing prothrombin was subjected to 20 mM Tris
CaCl ₂ concentration gradient of 0~0.1M in buffer of -HCl / 0.2M NaCl (pH7.5) was eluted by linearly load. The elution pattern of the total protein, the elution pattern of prothrombin, and the CaCl ₂ concentration gradient indicated by the A280 value are as shown in FIG. Prothrombin is eluted by low concentrations of calcium chloride,
It was separated from contaminating proteins with high adsorption characteristics to anion exchange resin. The purification efficiency by this chromatography step was extremely higher than that of Comparative Example 1.

Example 3 The prothrombin complex obtained in Example 1 was added to 25 mM
Q-Sepharose dialyzed against Na ₃ citrate / 0.2 M NaCl (pH 6.0) buffer and equilibrated with the same buffer
After adsorbing to the FF column, the column was washed to remove unbound proteins. 20 mM Tris-HCl
After replacement with a buffer solution of 0.2 M NaCl (pH 7.5), the fraction containing prothrombin was subjected to 20 mM Tris-H.
Cl / 0.2 M NaCl / 10 mM CaCl ₂ (pH 7.
Eluted in 5). The recovery rate of prothrombin by column chromatography, the purity (specific activity) of the obtained prothrombin, and the trypsin inhibitory activity value present in the obtained prothrombin fraction are as shown in Table 2.
As is clear from comparison with Table 1 showing the results of Comparative Example 2,
By the purification method according to the present invention, prothrombin with significantly improved purity compared to the conventional method was obtained, and it was possible to remove the trypsin inhibitory activity to below the measurement limit.

[0020]

[Table 2]

Example 4 Prothrombin (containing factor X) prepared according to Example 3 was concentrated using an ultrafiltration membrane to obtain calcium chloride,
PEG 4,000 and glycerol were added at a final concentration of 5 mM, 14% and 20%, respectively, and treated at 25 ° C. for 7 days. The obtained thrombin showed an activation rate of 88.4 (NIH units / prothrombin 1 plasma unit), and the specific activity was 529 NIH units / A280.

Example 5 The thrombin obtained in Example 4 was replaced with 20 mM Tris-HC
After adsorbing to SP-TOYOPEARL equilibrated with a 1 / 0.15 M NaCl (pH 8.0) buffer, washing was performed to remove unbound proteins. The thrombin fraction was converted to 20 mM N
Elution with a ₃ buffer citrate / 1.0M NaCl (pH7.0), concentrated, diafiltered, were subjected to freeze-drying and dry heat treatment. The specific activity of the obtained thrombin is 1,
It was 100 NIH units / A280.

[Brief description of the drawings]

FIG. 1 is a view showing an elution pattern when prothrombin is eluted from a Q-Sepharose FF column by a sodium chloride concentration gradient.

FIG. 2 is a view showing an elution pattern when prothrombin is eluted from a Q-Sepharose FF column by applying a calcium chloride concentration gradient in the presence of 0.2 M sodium chloride.

Claims (11)

[Claims] The present invention is characterized in that a solution containing prothrombin (sometimes referred to as blood coagulation factor II) is developed on an anion exchange resin to adsorb prothrombin, and prothrombin is eluted with a buffer containing a calcium salt. A method for purifying prothrombin. 2. The method for purifying prothrombin according to claim 1, wherein the concentration of the calcium salt is 100 mM or less. 3. The concentration of the calcium salt is 1 to 20 mM.
The method for purifying prothrombin according to claim 2, wherein 4. The method according to claim 1, wherein the calcium salt is calcium chloride,
The method for purifying prothrombin according to any one of claims 1 to 3, wherein the method is selected from calcium hydroxide and calcium acetate. 5. An ionic strength of a solution containing prothrombin is set to an ionic strength at which prothrombin can be adsorbed to an anion exchange resin in the absence of a calcium salt.
A method for purifying prothrombin, which further comprises adjusting a composition containing a calcium salt, and then developing the composition on the anion exchange resin to allow prothrombin to pass through. 6. The method for purifying prothrombin according to claim 5, wherein the ionic strength of the solution containing prothrombin in the absence of the calcium salt is 0.2 M or less in terms of sodium chloride concentration. 7. The method for purifying prothrombin according to claim 5, wherein the concentration of the calcium salt is 100 mM or less. 8. The concentration of the calcium salt is 1 to 20 mM.
The method for purifying prothrombin according to claim 7, wherein 9. The method according to claim 9, wherein the calcium salt is calcium chloride,
The method for purifying prothrombin according to any one of claims 5 to 8, wherein the method is selected from calcium hydroxide and calcium acetate. 10. A method for producing prothrombin and / or thrombin, comprising a step based on the purification method according to any one of claims 1 to 9. 11. A prothrombin and / or thrombin composition obtained by the production method according to claim 10.