Purification and activation method of prothrombin

Abstract

The invention discloses a method for purifying and activating prothrombin, which comprises the following steps: by taking cryoprecipitate-removed plasma as a raw material, washing and eluting by adopting an anion exchange resin adsorption method to obtain a solution containing the prothrombin and a blood coagulation factor X (FX), carrying out ultrafiltration on the solution to reduce the conductivity to be below 2.5 ms / cm, and concentrating the concentration of the prothrombin to be above 15IU / ml to obtain the prothrombin. And adjusting the concentration of FX to 50%-150% of the concentration of prothrombin. Adjusting the concentration of the prothrombin, adding a calcium chloride solution into the prothrombin until the final concentration of calcium chloride in the solution is 20-60mmol / l, uniformly stirring and mixing, and activating for 6-15 days at the temperature of 0-8 DEG C to obtain a thrombin solution. According to the method, the prothrombin recovery rate is high, the activation effect is good, and each IU of prothrombin can be activated to obtain 207.5 IU of thrombin titer. The problem that the thrombin titer recovery rate is not high is solved, and the yield of unit plasma thrombin titer is greatly increased.

Description

Technical Field

[0001] The present invention relates to the field of blood products, and in particular to a method for purifying and activating prothrombin. The method can be applied to the activation and subsequent purification preparation of human thrombin, as well as the activation and purification preparation of animal prothrombin, and can greatly improve the thrombin yield per unit plasma. Background Art

[0002] Prothrombin is one of the vitamin K-dependent factors synthesized by the liver. It is a single-chain glycoprotein with 579 amino acid residues and a molecular weight of 72KD. From the N-terminus, there is a Gla region (1-40), two loop regions (41-271) and a catalytic region (271-579). The Gla region contains 10 r-carboxyglutamic acid residues, and its main function is to bind to phospholipids by binding calcium ions. Loop region 1 participates in its interaction with substrates and cofactors, and loop region 2 can bind to FⅤa and contains the action site of FⅩa, histidine 205 to arginine 220. The catalytic region includes an activation region and a serine protease region. With the participation of calcium ions, FⅤa and phospholipids, prothrombin is activated by FⅩa. Prothrombin is cleaved at arginine 320 to generate an intermediate product, which is further cleaved at arginine 284 and arginine 155 to generate thrombin and prothrombin fragment 1 and prothrombin fragment 2. Thrombin is composed of A chain and B chain linked by disulfide bonds, containing 295 amino acid residues and a molecular weight of 36KD. The A chain contains 36 amino acid residues, also known as the light chain, and its function is unknown. The B chain contains 259 amino acid residues, also known as the heavy chain, which is the site of enzyme activity. The serine protease in the catalytic region of prothrombin is in the B chain. The serine protease region has protease activity and contains a site for recognizing and cleaving substrates. The enzyme active amino acids are histidine 363, aspartic acid 419 and serine 525. The arginine 382-arginine 393 fragment in this region is called the anion binding site, which is the site where prothrombin interacts with fibrinogen, thrombomodulin and hirudin.

[0003] Thrombin can convert fibrinogen into fibrin and also has the following functions: inducing platelet aggregation; activating factor XIII; converting plasminogen into plasmin, thereby activating the fibrinolytic system; activating fibrinolytic inhibitors activated by thrombin; activating factors V, VIII, and XI to generate more thrombin; activating the protein C system; stimulating wound healing, etc. As a hemostatic drug, thrombin lyophilized powder is available on the market at home and abroad, and is mainly suitable for hemostasis of small blood vessels, capillaries, and solid organ bleeding that are difficult to ligate. It is used for hemostasis of bleeding in trauma, surgery, oral, ear, nose, throat, urology, burns, orthopedics, neurosurgery, ophthalmology, obstetrics and gynecology, and digestive tract. In recent years, due to the advancement of medical technology, there are abundant alternative options. At present, the thrombin sold in China is mainly animal-derived. Because thrombin is cheap, thrombin is often listed as a short supply drug.

[0004] Thrombin was purified as early as the 1940s, and the system was separated from prothrombin by saturated ammonium sulfate precipitation. In the 1960s and 1970s, oxalate was mainly added to obtain prothrombin by isoelectric precipitation method, barium citrate adsorption method, etc., and then prothrombin was activated to thrombin with calcium ions or other activators (citrate). Although this type of method for extracting and purifying thrombin from blood plasma is simple and easy to operate, the output and specific activity are all low, and after extracting prothrombin, blood plasma is discarded, which is only suitable for blood plasma from animal sources. Entering the 1990s and the early 21st century, some new methods for extracting and purifying thrombin appeared, including ion exchange chromatography and affinity chromatography, etc., although thrombin with higher purity and activity can be obtained, the process is complicated, the time is long, and the production cost is high.

[0005] US PATENT 5907032 reported a method for producing thrombin using cryoprecipitated plasma as raw material. The eluate containing the coagulation factor complex was obtained by DEAE-cellose adsorption method, and then 70mmol / l calcium ions were added to the eluate. The pH was controlled at 6.4-6.6, and a large amount of thrombin was generated by incubation at 20°C for 18 hours. US PATENT 5714370 also introduced a process for producing thrombin using calcium ion activation. It used a calcium ion concentration of 2.5mmol / l. After activation for 80 minutes at pH 7.0 and 30°C, 1IU of prothrombin could produce 48IU of thrombin titer. In addition, Chinese patent CN02117151.3 discloses a method for purifying and activating the production of thrombin, extracting and purifying 70,000 IU of thrombin from 8kg of component III, and converting 800,000 IU of thrombin per ton of plasma. Because the prothrombin titer content in its initial component is relatively low, the final thrombin titer recovery is significantly lower. Chinese patent CN201610614283.8 also discloses a method for separating and activating purified thrombin from plasma components, and the thrombin recovery per ton of plasma has a certain improvement of up to 5-10 million IU. The main disadvantage of the above thrombin preparation method is that the prothrombin recovery rate is not high, the prothrombin activation efficiency after recovery is not high, and the prothrombin activation process is an exponentially amplified process, and the above two steps directly determine the yield of unit plasma thrombin titer.

[0006] In view of the above-mentioned shortcomings of the prior art, the present invention provides a method for extracting activated prothrombin from mixed blood. This method is simple to operate and can greatly improve the thrombin yield, thereby significantly reducing the unit thrombin production cost.

[0007] To achieve the above objectives, the present invention provides a method for extracting and activating prothrombin, comprising the following steps:

[0008] 1) adsorbing the cryoprecipitated plasma with an anion exchange resin, washing and eluting, and obtaining an eluate which is a purified prothrombin solution;

[0009] 2) ultrafiltration and concentration of the prothrombin solution obtained in step 1) using an ultrafiltration membrane to adjust the concentrations of prothrombin and coagulation factor X in the solution;

[0010] 3) The pH of the solution obtained in step 2) is adjusted with hydrochloric acid, and an activator is added thereto after sterilization filtration, and the mixture is mixed and activated.

[0011] In detail, the anion exchange resin in step 1) is DEAE Sephadex-A50, and the dry weight of DEAE Sephadex-A50 ion exchange resin added per ton of plasma can be 0.5-2.0 kg. The ion exchange resin washing liquid used contains sodium citrate and sodium chloride, wherein the content of sodium citrate can be 0.15-0.25 mol / l, the content of sodium chloride can be 0.05-0.1 mol / l, and the pH value is 6.5-7.5. The eluent contains sodium citrate and sodium chloride, wherein the content of sodium citrate can be 0.15-0.25 mol / l, the content of sodium chloride can be 0.5-1.5 mol / l, and the pH value is 6.5-7.5. The eluent obtained per ton of plasma ranges from 50 to 100 L.

[0012] In the specific implementation, the ultrafiltration membrane cutoff pore size in step 2) is 5KD or 10KD or 30KD, the ultrafiltration buffer used is water for injection, the temperature of the water for injection used is less than or equal to 25°C, and the conductivity value of the final solution after ultrafiltration is 0.01-2.50ms / cm. In the specific implementation, the prothrombin titer in the prothrombin solution after ultrafiltration is controlled at 15-30IU / ml. This solution can be immediately used for the next step or can be frozen and temporarily stored in a -20--30°C cold storage or freezer.

[0013] In the specific implementation, when the ultrafiltration prothrombin solution in step 2) is activated by adding an activator, it can be directly adjusted with cold water for injection so that the final concentration of prothrombin therein is in the range of 5 to 15 IU / ml, and the content of coagulation factor X therein is 50% to 150% of the content of prothrombin, and the optimal concentration content is 100%;

[0014] In the specific implementation, the hydrochloric acid concentration range for adjusting the pH in step 3) is 0.1-0.5 mol / L, and the final pH concentration range of the adjusted solution is 6.5-8.5, with the optimal pH value being 7.5. After the pH value is adjusted, sterile filtration should be performed, and during sterile filtration, the prothrombin solution and the activator solution need to be sterile filtered separately.

[0015] In the specific implementation, the prothrombin activator in step 3) is a 1 mol / l calcium chloride solution, and sterilization filtration should be performed when added to the prothrombin solution to be activated. The final calcium ion concentration in the prothrombin solution to be activated is in the range of 20 to 60 mmol / l, and the optimal concentration is 40 mmol / l.

[0016] In the specific implementation, the temperature range of the prothrombin activation in step 3) is 0-10° C. The incubation activation process can be carried out with stirring or under static conditions, and the incubation time is 6-15 days.

[0017] As described above, the prothrombin purification and extraction efficiency of the present invention is high, and the prothrombin yield in the DEAE Sephadex-A50 adsorption step can reach more than 90% of the theoretical recovery rate; the prothrombin activation efficiency is high, and each IU of prothrombin can be activated to obtain 207.5 IU of thrombin titer, and each ton of plasma can activate a maximum of 167 million IU of thrombin titer. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The effect diagram of prothrombin activation under different initial pH conditions in Example 2 of the present invention Specific embodiments

[0019] Example 1

[0020] 1) 1000 kg of cryoprecipitated plasma was prepared by centrifugation and pumped into an adsorption tank, into which the treated DEAE Sephadex-A50 ion exchange resin was added, the dry weight of the resin was 1.0 kg, and the resin was stirred for 30 minutes of adsorption, and then the DEAE Sephadex-A50 ion exchange resin was separated by filtration, and then washed and eluted. The washing liquid had a sodium citrate content of 0.2 mol / l, a sodium chloride content of 0.09 mol / l, a pH value of 7.1, and an eluent had a sodium citrate content of 0.2 mol / l, a sodium chloride content of 1.0 mol / l, and a pH value of 7.1. A total of 90.5 kg of eluent was obtained. After testing, the total titer of the recovered prothrombin was 832,600 IU, and the total titer of FX was 891,300 IU.

[0021] 2) The eluate in step 1) was ultrafiltered and concentrated with cold water for injection, the pore size of the ultrafiltration membrane was 10KD, and the equal volume was ultrafiltered 7 times, the weight of the ultrafiltered solution was 50kg, the conductivity of the ultrafiltered solution was 0.8ms / cm, and the pH was 8.4. The ultrafiltered solution was sampled to detect the titers of prothrombin and coagulation factor X and the total amount recovered was calculated, and the total titer of prothrombin was measured to be 832,000 IU, and the total titer of recovered FX was 852,300 IU.

[0022] 3) Take 30 kg of the ultrafiltration solution in step 2), dilute it to 50 kg with cold water for injection, adjust the pH to 7.5 with 0.25 mol / l hydrochloric acid, and then filter it into the sterilized activation tank with a 0.22 um filter element, and pump 2.5 kg of 1 mol / l calcium chloride solution after sterilization and filtration into the activation tank. After stirring evenly, cool it to 4 ° C and start incubation activation. After about 9 days of activation, stop incubation, take out the activated solution to complete the subsequent steps of thrombin purification and production.

[0023] After 9 days of activation, the thrombin titer was detected by sampling and testing to be 1683 IU / ml, namely, after activation of 30 kg of ultrafiltrated prothrombin solution, a total of 88.36 million IU of thrombin titer was obtained. After conversion, each IU of prothrombin can be activated to obtain 177.4 IU of thrombin titer, and each ton of plasma can be activated to obtain approximately 147 million IU of thrombin titer.

[0024] Example 2

[0025] 1) Take 1 kg of the ultrafiltered prothrombin solution obtained in Example 1, divide it into five equal parts, and dilute each part to 330 ml with cold water for injection;

[0026] 2) Using 0.25 mol / l hydrochloric acid, adjust the pH of the five prothrombin solutions in step 1) to 6.85, 7.25, 7.45, 7.65, and 7.85, respectively, and then sterilize and filter into 500 ml serum bottles, numbered 1#, 2#, 3#, 4#, and 5#;

[0027] 3) Add 16 ml of sterilized 1 mol / l calcium chloride solution to each bottle in step 2), shake well and incubate at 2-8°C for 14 days;

[0028] 4) Samples were taken on the 6th, 9th, 12th and 14th days to test the thrombin titer. The activation results are shown in the attached Figure 1 According to calculation, under the conditions of this method, each IU of prothrombin can activate a maximum thrombin titer of 207.5 IU.

Claims

1. The present invention relates to a method for extracting and activating prothrombin, comprising the following steps: 1) adsorbing the cryoprecipitated plasma with an anion exchange resin, washing and eluting, and obtaining an eluate which is a purified prothrombin solution; 2) ultrafiltration and concentration of the prothrombin solution obtained in step 1) using an ultrafiltration membrane to adjust the concentrations of prothrombin and coagulation factor X in the solution; 3) The pH of the solution obtained in step 2) is adjusted with hydrochloric acid, and an activator is added thereto after sterilization filtration, and the mixture is mixed and activated.

2. According to the method for extracting prothrombin as claimed in claim 1, the anion exchange resin in step 1) is DEAE Sephadex-A50, and the eluate obtained per ton of plasma ranges from 50 to 100 L.

3. According to the prothrombin extraction method of claim 1, the ultrafiltration membrane cutoff pore size in step 2) is 5KD or 10KD or 30KD, the ultrafiltration buffer used is water for injection, the temperature of the water for injection used is not higher than 25°C, and the conductivity value of the final solution after ultrafiltration is 0.01-2.50ms / cm.

4. The method for extracting prothrombin according to claim 1, wherein the final concentration of prothrombin in the prothrombin solution after ultrafiltration in step 2) ranges from 5 to 15 IU / ml, and the optimal concentration is 10 IU / ml.

5. The method for extracting prothrombin according to claim 1, wherein the prothrombin solution after ultrafiltration in step 2) contains, in addition to prothrombin, also coagulation factor X, the content of which is 50% to 150% of the content of prothrombin, and the optimal concentration is 100%.

6. The prothrombin activation method according to claim 1, wherein the concentration of hydrochloric acid used to adjust the pH in step 3) is in the range of 0.1 to 0.5 mol / L; the final pH concentration of the solution after adjustment is in the range of 6.5 to 8.5, and the optimal pH value is 7.

5.

7. The prothrombin activation method according to claim 1, wherein in the sterilization filtration step in step 3), the prothrombin solution and the activator solution need to be sterilized and filtered separately.

8. The prothrombin activation method according to claim 1, wherein the prothrombin activator in step 3) is a 1 mol / l calcium chloride solution, and the final calcium ion concentration of the prothrombin solution to be activated ranges from 20 to 60 mmol / l, with an optimal concentration of 40 mmol / l.

9. The method for activating prothrombin according to claim 1, wherein the temperature range of the prothrombin activation in step 3) is 0 to 10°C, and the activation time range is 6 to 15 days.

10. The plasma according to claim 1 can be human plasma or plasma from animals such as pigs or cows.

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