KR890002070B1 - Process for preparing thrombins - Google Patents

Abstract

50 Liter of bovine serum was treated with 2kg BaSO4 and stood overnight at 4≰C. The resulting precipitate was treated with Nacitrate contg. 0.45% NaCl and centrifuged continuously at 2,000 rpm to give barium sulfate cake. After concentrating by dialysis or ultrafiltration, the concentrate was mixed with 1M Tris-HCl (pH 7.2) buffer, 1M CaCl2 and 6M NaCl. The soln. was stirred with thromboplastin for activation of thrombin, centrifuged at 8,000 rpm and trated with 490g (NH4)2SO4 to the filtrate to give a product.

Description

Method of manufacturing thrombin

The present invention relates to a method for producing thrombin having excellent efficacy as a hemostatic agent.

Preventing blood loss during trauma, surgery, hemophilia or other bleeding is a very important issue.

The clotting process of blood exposed outside the blood vessel occurs through a very complex plotting mechanism. In this mechanism, several protein elements act as enzymes or coenzymes, and Ca ⁺ ions, phospholipids, and thromboprastin are known to be required. As a final step, some peptides are separated from prothrombin to form thrombin. This thrombin is dissolved in the blood and acts on the most fibrinogen as a protein to remove some of the peptides at the C-terminus of the fibrinogen to form a fibrin monolith. A crot is formed to coagulate blood and hemostasis.

There are several types of hemostatic agents currently used, but a good one is thrombin. Thrombin formulations have been used for a long time, but they are not widely used because of their high complexity and high manufacturing cost.

For example, in US Patent No. 2,398,077, potassium oxalate solution is added to bovine blood to remove blood cells and centrifuged, and about 10-fold distilled water is added to the plasma obtained by centrifugation. Magnesium hydroxide was added to the obtained plasma and water was added thereto, followed by placing in a pressurized vessel, and carbon dioxide gas was added under pressure to obtain prothrombin. The dialysis was removed to remove magnesium ions, and the pH was adjusted to 5.3 to add calcium salt to the prothrombin solution. Thrombin is obtained by obtaining an activated thrombin solution from which thrombin has been removed, and adding the organic solvent which is miscible with water thereto, dissolving the precipitate in water, and adding the organic solvent to the obtained supernatant and vacuum drying.

However, this method has to go through several tens of steps, and moreover, even through this complicated process, it is difficult to obtain pure thrombin, and the yield is extremely low, and a large quantity of thrombin products cannot be obtained.

The present inventors have conducted a long study to solve this problem, and found that the addition of barium sulfate to bovine hydroxide plasma, adsorbing and eluting with sodium citrate, can produce thrombin with high purity and high yield with a simple operation. The invention was completed. The object of the present invention therefore relates to a novel and improved process for producing thrombin formulations.

The present invention is described in detail as follows.

Stage 1, Separation of Plasma from Bovine Blood: Exposing the blood of a bovine out of blood vessels immediately initiates a coagulation mechanism by the Intrinsic Pathway, which causes prothrombin to turn into thrombin, resulting in a clogged mass of blood. As soon as the blood of a cow is taken from a dead cow, it must be mixed with a solution of sodium hydroxide, a chelating agent. Take blood from cattle within 1 to 2 minutes of death and mix well with 2.5% sodium hydroxide solution in 9 volumes of blood (V / V). At this time, it is good to use a stainless container and stir slowly while receiving the blood and mix well with sodium hydroxide solution. Oxalate treated with oxalate is removed from blood cells using a serial centrifuge to obtain plasma. At this time, about half of the skin is obtained in plasma.

Adsorption and elution on barium sulphate in step 2, stir while slowly adding 25-40 g of BaSo ₄ per liter to the plasma obtained in step 1. Add BaSo ₄ and stir slowly using a motor-stirred stirrer. At this time, the temperature is carried out under 4 ℃, it is good to avoid the foaming. The BaSo ₄ has prompted thrombin, factor VII, factor IX, factor X has been adsorbed the protein other than such value is not adsorbed on BaSo _4. The BaSo ₄ mixed carbon solution thus obtained was stirred for a certain time, and left in a cool place for several hours to sink BaSo ₄ adsorbed with various proteins. The BaSo ₄ precipitate thus obtained was clouded with stirring by adding 0.45% NaCl (W / v) sodium citrate solution to a final concentration of 1 mM and washing the BaSo ₄ precipitate. The BaSo ₄ precipitate is then recovered using a continuous centrifuge and the washing mentioned above is repeated 2-3 times. Re-collect in washed BaSo ₄ and add 100 mM sodium citrate and stir in suspension with BaSo ₄ for a suitable time. This causes the protein attached to BaSo ₄ to be eluted into solution, with prothrombin also eluting into solution. The suspension is centrifuged to recover a solution of prothrombin and the like. At this time, BaSo ₄ should be removed.

Step 3, Activate thrombin of prothrombin: Lower the high concentration of citrate ions in the solution obtained in step 2 below 10 ^-3 M using dialysis or ultrafiltration. The elution solution with reduced citric acid ion concentration was changed to 20 mM Tris solution (containing 150 mM NaCl and 20 mM CaCl ₂ ) to pH 7.2, followed by addition of thromboprastin isolated from bovine brain to activate prothrombin with thrombin. At this time, the higher the temperature, the better the activation, but the protein is left in the stirrer attached to the motor for a long time at a low temperature to stabilize.

Step 4 Fractionation of thrombin with ammonium sulfate: In step 3, the precipitates are removed from the solution from the activated prothrombin to thrombin by centrifugation. Crushed (NH ₄ ) ₂ SO ₄ was added to the removed solution with gentle stirring to 40% saturation, and then left to cool for an appropriate time. The precipitated solution is centrifuged to remove the precipitate, and the resulting solution is again added with (NH ₄ ) ₂ SO ₄ to reach 75% saturation, left to stand again, and the precipitate is recovered by centrifugation. The recovered precipitate is dissolved in 50mM glycine and 20mM CaCl ₂ solution and dialyzed using a dialysis membrane. Glycine and CaCl ₂ are added to the solution so that the ratio between protein and salt is adjusted.

Step 5, Sterilization and lyophilization: Place the above solution in a dish or bowl of low height, wide bottom and reflective bottom surface and irradiate for 30 minutes under sterile UV light. At this time, the depth of the contained solution should be about 1cm. Then, permerol (Phermerol ^R : benzotonium chloride) is dissolved in a small amount of distilled water, and sterilized at high temperature and high pressure. After mixing a certain ratio (protein: Phermerol = 100: 1), a suitable amount of thrombin in a pre-sterilized vial is added. The solution is divided and lyophilized under aseptic conditions.

Next, the present invention will be described in detail by way of examples.

EXAMPLE

Separation of Plasma from Bovine Blood: Add 10 l of 2.5% sodium hydroxide to 90 l of fresh bovine blood and stir well. Centrifugation at RPM 5000 removes the blood cells and yields approximately 50 L of plasma.

2. Adsorption and elution by barium sulphate: 2 kg of barium sulphate is added to the plasma obtained in the first step, and the mixture is stirred slowly with a magnetic stirrer for 30 minutes to obtain a suspension.

The suspension is left at 4 ° C. overnight and then the supernatant is removed. Sodium citrate solution containing 0.45% sodium chloride was added to the barium sulfate from which the supernatant was removed to adjust the pH to 7.2 mM sodium citrate. The pH was adjusted to 7.2. The mixture was stirred slowly for 30 minutes and continuously centrifuged at RPM 2000 to prepare the barium sulfate cake. Get

The barium sulfate cake is washed twice with 1 mM sodium citrate solution containing 0.45% sodium chloride.

0.1 M sodium citrate was added to the obtained barium sulphate solution to adjust the pH to 5.8 by adding about 1/20 of the total plasma (V / V), followed by stirring for 30 minutes with a magnetic stirrer to obtain a suspension.

Centrifuge for 30 minutes at 4 ° C. at 2000 rpm to collect supernatant and repeat the above procedure twice to obtain an eluate and combine with the supernatant.

3. Activation with thrombin of prothrombin: The citrate ions remaining in the solution obtained in step 2 are subjected to dialysis or ultrafiltration to reduce the concentration of citrate ions to 10 ⁻³ M or less, and then the dialysis solution is concentrated to about 2 L. 40 ml of 1 M Tris-HCl (pH 7.2), 40 ml of 1 M CaCl ₂ , and 50 ml of 6 M NaCl were added to the concentrate, and a small amount of thromboprastin extracted from bovine brain was added and stirred slowly. Incubate at 4 ° C. for 15 hours to activate thrombin for prothrombin.

4. Fractionation of thrombin with ammonium active acid: The activated thrombin solution obtained in step 3 was centrifuged at 8000 rpm at 4 ° C. to remove the precipitate, and slowly added 490 g of ammonium sulfate and stirred with a magnetic stirrer at 4 ° C. for 2 hours. do.

The precipitate was obtained by centrifugation at 5000 rpm for 30 minutes, and 550 g of ammonium sulfate was added to the filtrate and stirred for 5 hours, followed by centrifugation at 8000 rpm for 30 minutes to obtain a precipitate. The precipitates are combined and dissolved in 200 ml of 50 mM glycine containing 10 mM CaCl ₂ (pH 7.2).

After dialysis this liquid with 25 times of distilled water, dialysis is repeated 3 times. After the dialysis, an appropriate amount of glycine and CaCl ₂ is added to adjust the salt ratio to 2: 1 (W / W).

5. Sterilization and freeze drying: Put the solution obtained in step 4 into a container with a large surface and reflect the surface and irradiate with UV etc. for 30 minutes.

Here, the permerol (benzotonium chloride) is added (1 mg per 100 mg of protein) and sterilized for 15 minutes at 15 psi pressure at 121 ° C., filled in vials, and lyophilized to prepare a product.

Claims (1)

Sodium hydroxide was added to bovine blood and barium sulfate was added to adsorbed plasma to adsorb prothrombin, eluted with sodium citrate, and thromboprastin extracted from bovine brain was added to activate prothrombin with thrombin and fractionated with ammonium sulfate. How to make thrombin.