JPH0219400A - Method for producing thrombin or prothrombin - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a method for producing thrombin or prothrombin derived from human plasma.

[Prior art]

Thrombin is a serine protease with a molecular weight of 39,000, and is a proteolytic enzyme that acts in the final step of the blood coagulation process. That is, it is a proteolytic enzyme that produces blood coagulation by acting on fibrinogen and producing fibrin.

For this reason, thrombin is clinically used as a local hemostatic agent in the surgical field and as a hemostatic agent for upper gastrointestinal bleeding in the internal medicine field.

Incidentally, this thrombin is prepared by treating prothrombin extracted from human plasma with thromboplastin in the presence of Ca ions, and is formulated by subjecting it to treatments such as sterilization and freeze-drying.

Thrombin is usually (I) prothrombin sperm L(n)
It is produced through the following steps: conversion of prothrombin to thrombin, and (■) purification of thrombin.

Among these, regarding the purification of prothrombin (I),
Methods utilizing adsorption to inorganic salts, methods using anion exchangers, methods using affinity chromatography, and the like are known, for example, from the following publications.

・(J, Biol, Chell,, 174.56
5 (1954), Sieve, J., Physiol.
172.731 (1953)・Biochemistry, View (12),
890-901 (1968)・Special public service 1977-502
Publication No. 02/J, Biol, Chell, Gateplate 2857
(1959) ) By the way, in conventional methods, human plasma itself or Cohn's 100% (Japanese Patent Application No. 56-500569), etc., have been used as the starting material.

However, when plasma itself is used, the purification efficiency of thrombin is not good, and when Cohn's fraction (2) is used, the preparation of fraction (2) itself requires complicated operations, resulting in high cost and non-conformity. There are problems such as being industrial.

[Problem to be solved by the invention]

In view of the above-mentioned circumstances, the present inventors have conducted various studies and found that it is possible to produce prothrombin or thrombin using Cohn's fraction molecules +n0m.

Furthermore, it has been found that since fibrinogen is contained in both parts t+n+m of the cone, gelation occurs if left as is, making the operation for producing thrombin difficult.

Therefore, in order to solve these problems, the present inventors
As a result of further intensive research, by treating the corn fraction i+n+m, which is the starting material, with colloidal silicic acid,
It has been found that the purification efficiency is increased, the operation is simple, and the cost can be reduced, so that it is industrially applicable.

[Means to solve the problem]

The present invention has been completed based on the above new findings, and has the following gist (1) to (3).

(1) A method for producing thrombin or prothrombin, which comprises using Cohn's fraction ++nm derived from human plasma.

(2) Percentage of Cohn derived from human plasma 1+n+lII
The manufacturing method according to (1) above, which includes the steps of treating with colloidal silicic acid and collecting the non-adsorbed fraction.

(3) The production method according to (2) above, further comprising a step of treating with an anion exchanger and collecting an eluted fraction.

More specifically, the method for producing thrombin or prothrombin of the present invention can be roughly divided into (1) purification of prothrombin, (■) conversion of prothrombin to thrombin, (
I[[) I-consists of the purification of rhombin, in particular (1)
There are characteristics.

(1) Purification of prothrombin (i) Starting material As the starting material, Cohn's fraction I+■10■ derived from human plasma is used.

Both parts have a pH of about 6 to 9, preferably pH 6.5 to 9.
8.5, and a salt concentration of about 0.05 to 0.3 M, preferably about 0.1-0.2 M, in an appropriate solvent (e.g., sodium chloride solution, Tris-hydrochloric acid solution, ammonium acetate solution, sodium citrate). solution, etc.) to extract prothrombin. The amount of the solvent added is about 1 to 10 ffi, preferably about 5 to 10 ffi per kg of the two parts.

(it) The extracted fraction from the colloidal silicic acid treatment (i) is brought into contact with colloidal silicic acid and the non-adsorbed fraction is collected.

Examples of colloidal silicic acid include silica gel, light anhydrous silicic acid, diatomaceous earth, acid clay, bentonite, kaolin, and magnesium aluminate silicate. Light anhydrous silicic acid is preferably used.

For example, the contact conditions are as follows. That is, the amount of colloidal silicic acid per 100 volumes of extracted fraction is about 0.1 to 10 volumes, preferably about 0.5 to 5 volumes, and ρ■ is pH
The pH is about 6 to 9, preferably about 6.5 to 8.5, and the salt concentration is about 0.05 to 0.3M, preferably 0.1
It is about ~0.2M.

This treatment can be carried out by either a batch method or a column method, but preferably a batch method is used.

In the case of a batch method, it is carried out by mixing and stirring at 5 to 25°C for about 5 minutes to 1 hour.

The supernatant (non-adsorbed fraction) is then filtered or centrifuged.
Collect.

Further purification can also be performed using known prothrombin purification techniques. Specifically, the salting-out method (method using ammonium sulfate), adsorption method (method using barium sulfate, magnesium hydroxide, aluminum hydroxide, etc. as an adsorbent)
, fractionation method (method using acrinol, PEG, etc.), anion exchanger treatment, affinity chromatography treatment, etc., and anion exchanger treatment is particularly preferred. Therefore, next, the anion exchanger treatment step will be described.

(iii) Anion exchanger treatment The non-adsorbed fraction obtained by the above colloidal silicic acid treatment is brought into contact with an anion exchanger, and the eluted fraction is collected as a prothrombin-containing fraction.

Anion exchange 1! As the A-body, DEAE series (for example,
DEAE-agarose, DEAE-dextran, DE
AE-cellulose, etc.), QAE-based (QAE-agarose, QAE-dextran, etc.), and the like.

The contact conditions are: pH about 6 to 8, preferably about ρ117, salt concentration about 0.05 to 0.2M, preferably 0.1 to
The elution conditions are ρ■6 to 8, preferably pH about 7, and salt concentration of about 0.2 to 0.5M, preferably about 0.2 to 0.3M.

Note that further purification can be performed using known prothrombin purification techniques.

(■) Conversion of prothrombin to thrombin (1)
A known method may be used to convert the prothrombin obtained in ) to thrombin.

Specifically, examples include a method in which thromboplastin, snake venom, etc. are made to act on prothrombin in the presence of Caz+.

(■) Purification of thrombin The thrombin obtained in the above (II) can be purified by any known method. That is, treatment with a cation exchanger,
Examples include ammonium sulfate fractionation treatment and affinity chromatography treatment.

As a cation exchanger, for example, Amberlite TR
C50, sulfoethyl-Sephadex, etc. are used [Biochem, 10.13.2501 (19
71)).

Affinity chromatography treatment includes, for example, a treatment method using a carrier in which sulfonate and arginyl methyl ester are bonded to polystyrene, an adsorbent with heparin Na as a ligand, etc. (J,
Chromatography+ 3639540
0 (1986), Thrombosis Re5e
Arch, 11.799-808 (1977)).

In addition, further purification can be performed by known means such as dialysis, ultrafiltration, and gel filtration.

The specific activity of the purified thrombin thus obtained was 10
The amount is preferably about 0 to 1000 units/■ protein.

The thrombin thus obtained is a fraction of Cohn's molecules +n+
Various proteins that contaminate nr (e.g., fibrinogen,
Fibrin, α1 globulin, α2 globulin, β globulin, T globulin, protease, etc.) are substantially removed.

The thrombin is formulated by a method known per se.

That is, treatments such as addition of excipients, heating, sterilization, sterilization filtration, dispensing and aliquoting, and freeze drying are performed as necessary.

[Effects] According to the production method of the present invention, thrombin can be efficiently purified with simpler operations than in conventional production methods, and the cost can be sufficiently reduced.

Therefore, the method of the present invention is a useful method suitable for industrial scale.

[Examples] Examples are given below to explain the present invention in more detail, but the present invention is not limited by these in any way.

Example Cohn's fraction derived from human plasma [+II+n 0.15 M sodium chloride 61 per 1 kg was added and suspended. After collecting the supernatant, light silicic anhydride (Aerosil 380, manufactured by Nippon Aerosil Co., Ltd.) was added to lv/v%. and mixed for 10 to 60 minutes at room temperature.

The filtrate was collected by filtration and mixed with DEAE-agarose (DEAE-Sepharose Fast F1a, 7111797 vM) which had been equilibrated with 0.01M citrate buffer (pH 7) containing 0.125M sodium chloride.
) was added. After washing with the above buffer, use citrate buffer 1 (pl+7) containing 0.25M sodium chloride. lLt,
Prothrombin was purified.

Thromboplastin prepared from human placenta (1/10 volume, human plasma (11/40 volume) and calcium chloride solution (2%) volume at 11/10 volume) was added to this prothrombin.
Crude thrombin (10 units of thrombin activity per mg protein) was obtained by thrombin conversion. The crude thrombin was purified using sulfopropyl (SP)-Sephadex column chromatography method [Lundblad, R.
L., Biochemistry, 10.2501
(1971)], and after concentrating the purified thrombin, 10
Dialysis was performed against 0 mM citrate buffer (pH 7.0) to prepare a thrombin solution (3500 units/form, thrombin activity 500 units per mg protein).

Thrombin? Add 8 g of rice bran and 4.2 g of arginine per 15 d of volume, and adjust the pH to 6 with sodium hydroxide.
．． 2, 8 laps were made. This mixture was heat treated at 60°C for 10 hours and then dialyzed against the above buffer.

This thrombin solution was sterilized and filtered, and then freeze-dried to obtain thrombin powder.

By the above-described treatment, purified thrombin of approximately 300,000 units per kg was recovered.

Experimental Examples Using prothrombin-containing fractions obtained at each stage in the example (supernatant collection, light silicic anhydride treatment, DEAE-agarose treatment), the presence or absence of gelation and the conversion efficiency to thrombin were investigated. .

The presence or absence of gelation was visually observed after being left at 4°C for 2 hours.

The conversion efficiency to thrombin indicates the degree of conversion of prothrombin to thrombin, and the conversion operation was performed according to the example, and the prothrombin activity concentration before conversion and the thrombin activity concentration after conversion were measured. , was obtained by calculating the ratio. The results are shown in Table 1.

[Extra white]

As shown in Table 1, light silicic anhydride treatment does not cause gelation and also improves conversion efficiency. Further DE
It was found that AE-agarose treatment further improved the conversion efficiency.

Claims (3)

[Claims] (1) A method for producing thrombin or prothrombin, which comprises using Cohn's fractions I + II + III derived from human plasma. 2. The production method according to claim 1, which comprises the steps of: (2) treating Cohn's fractions I+II+III derived from human plasma with colloidal silicic acid and collecting the non-adsorbed fraction. (3) The manufacturing method according to claim (2), further comprising the step of treating with an anion exchanger and collecting an eluted fraction.