Preparation method of thrombin

Abstract

The invention belongs to the technical field of biological medicine grafting, and provides a thrombin preparation method which comprises the following steps: adding an anticoagulant into pig plasma, standing, filtering and centrifuging to obtain supernate a; carrying out virus inactivation on the supernatant a by adopting an S / D method, namely adding tributyl phosphate and tween-8 into the supernatant a to obtain a solution b; carrying out column chromatography on the solution b by adopting a DEAE-cellulose anion exchange column, and then eluting by using an eluent to obtain an eluent c; mixing the eluate c with a trisodium citrate aqueous solution, carrying out dialysis treatment, and then adjusting the pH value to obtain dialysate d; mixing the dialysate d with a calcium chloride solution, and activating to obtain a thrombin solution; sequentially performing ultrafiltration concentration and freeze drying on the thrombin solution to obtain the thrombin. According to the preparation method disclosed by the invention, more than 50,000 IU of thrombin products can be extracted from each liter of plasma, so that the extraction efficiency is greatly improved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a method for preparing thrombin. Background Art

[0002] Thrombin is an important biological enzyme widely used in the fields of medicine, pharmaceuticals and biotechnology. It is mainly used to treat local hemostasis and blood coagulation during certain surgeries. Traditional thrombin preparation methods usually involve complex process steps and high costs, and may also cause problems such as viral contamination and unstable product quality. Therefore, it is of great practical significance to develop an efficient, safe and cost-effective method for preparing thrombin. Summary of the invention

[0003] The purpose of the present invention is to provide a method for preparing thrombin to overcome the defects of the prior art.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The present invention provides a method for preparing thrombin, comprising the following steps:

[0006] (1) adding an anticoagulant to porcine plasma, allowing the plasma to stand, and then filtering and centrifuging to obtain a supernatant a;

[0007] (2) inactivating the virus in the supernatant a by using the S / D method, i.e., adding tributyl phosphate and Tween-8 to the supernatant a to obtain a solution b;

[0008] (3) performing column chromatography on solution b using a DEAE-cellulose anion exchange column, and then eluting with an eluent to obtain an eluate c;

[0009] (4) mixing the eluate c and a trisodium citrate aqueous solution for dialysis treatment, and then adjusting the pH to obtain a dialysate d;

[0010] (5) mixing the dialysate d and the calcium chloride solution for activation to obtain a thrombin solution;

[0011] (6) The thrombin solution is sequentially subjected to ultrafiltration concentration and freeze-drying to obtain the thrombin.

[0012] Preferably, the anticoagulant in step (1) is one of trisodium citrate solution or potassium oxalate solution;

[0013] The concentration of the anticoagulant is 0.1 to 0.15 mol / L;

[0014] The volume ratio of the anticoagulant to blood is 1:7 to 1:9.

[0015] Preferably, the standing time in step (1) is 3 to 5 hours;

[0016] The standing temperature is 5-15°C.

[0017] Preferably, the eluent in step (3) is one or both of trisodium citrate solution and amino acid solution;

[0018] The concentration of the eluent is 0.05-0.25 mol / L;

[0019] The pH of the eluent is 6-9.

[0020] Preferably, the elution flow rate in step (3) is 1 to 2 mL / min.

[0021] Preferably, the concentration of the trisodium citrate solution in step (4) is 0.01 to 0.1 mol / L;

[0022] The pH is 6.0 to 6.5.

[0023] Preferably, the temperature of the dialysis treatment in step (4) is 0 to 10°C;

[0024] The dialysis treatment time is 2 to 3 hours.

[0025] Preferably, the concentration of the calcium chloride solution in step (5) is 0.05 to 0.15 mol / L;

[0026] Preferably, the temperature of the activation treatment in step (5) is 15 to 25°C;

[0027] The activation treatment time is 1 to 3 hours.

[0028] Preferably, the ultrafiltration concentration time in step (6) is 4 to 6 hours.

[0029] Beneficial effects:

[0030] The invention provides a method for preparing thrombin. The method can extract more than 50,000 IU of thrombin product per liter of plasma, greatly improving the extraction efficiency. Only a few steps such as centrifugation, sample chromatography, ultrafiltration desalting, activation, ultrafiltration concentration and freeze drying are required. Industrial production can be easily realized, manual operation and errors are reduced, and an organic solvent purification method is abandoned. The method is harmless to personnel and the environment, and waste discharge meets environmental protection requirements, thereby reducing environmental pollution. DETAILED DESCRIPTION

[0031] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0032] The present invention provides a method for preparing thrombin, characterized in that it comprises the following steps:

[0033] (1) adding an anticoagulant to porcine plasma, allowing the plasma to stand, and then filtering and centrifuging to obtain a supernatant a;

[0034] (2) inactivating the virus in the supernatant a by using the S / D method, i.e., adding tributyl phosphate and Tween-8 to the supernatant a to obtain a solution b;

[0035] (3) performing column chromatography on solution b using a DEAE-cellulose anion exchange column, and then eluting with an eluent to obtain an eluate c;

[0036] (4) mixing the eluate c and a trisodium citrate aqueous solution for dialysis treatment, and then adjusting the pH to obtain a dialysate d;

[0037] (5) mixing the dialysate d and the calcium chloride solution for activation to obtain a thrombin solution;

[0038] (6) The thrombin solution is sequentially subjected to ultrafiltration concentration and freeze-drying to obtain the thrombin.

[0039] In the present invention, the anticoagulant in step (1) is preferably one of trisodium citrate solution or potassium oxalate solution.

[0040] In the present invention, the concentration of the anticoagulant is preferably 0.1 to 0.15 mol / L, more preferably 0.11 to 0.14 mol / L, and even more preferably 0.12 to 0.13 mol / L.

[0041] In the present invention, the volume ratio of the anticoagulant to blood is preferably 1:7 to 1:9, more preferably 1:7.2 to 1:8.8, and more preferably 1:7.5 to 1:8.5.

[0042] In the present invention, the standing time in step (1) is preferably 3 to 5 hours, more preferably 3.2 to 4.8 hours, and even more preferably 3.5 to 4.5 hours.

[0043] In the present invention, the standing temperature is preferably 5 to 15°C, more preferably 6 to 14°C, and even more preferably 7 to 13°C.

[0044] In the present invention, the S / D method for inactivating viruses in step (2) is a conventional method in the art, wherein an aqueous solution of tributyl phosphate with a volume ratio concentration of 0.3% and an aqueous solution of Tween-80 with a weight ratio concentration of 1% are mixed with the supernatant a and stirred.

[0045] In the present invention, the stirring time is preferably 3 to 6 hours, more preferably 3.2 to 5.8 hours, and more preferably 3.5 to 5.5 hours.

[0046] In the present invention, the eluent in step (3) is preferably one or both of trisodium citrate solution and amino acid solution.

[0047] In the present invention, the concentration of the eluent is preferably 0.05 to 0.25 mol / L, more preferably 0.07 to 0.23 mol / L, and even more preferably 0.1 to 0.2 mol / L.

[0048] In the present invention, the pH of the eluent is preferably 6 to 9, more preferably 6.2 to 8.8, and even more preferably 6.5 to 8.5.

[0049] In the present invention, the elution flow rate in step (3) is preferably 1 to 2 mL / min, more preferably 1.1 to 1.9 mL / min, and even more preferably 1.2 to 1.8 mL / min.

[0050] In the present invention, the concentration of the trisodium citrate solution in step (4) is preferably 0.01 to 0.1 mol / L, more preferably 0.02 to 0.09 mol / L, and even more preferably 0.03 to 0.08 mol / L.

[0051] In the present invention, the pH is preferably 6.0 to 6.5, more preferably 6.1 to 6.4, and even more preferably 6.2 to 6.3.

[0052] In the present invention, acetic acid or sodium bicarbonate can be used to adjust the pH in step (4).

[0053] In the present invention, the temperature of the dialysis treatment in step (4) is preferably 0 to 10°C, more preferably 1 to 9°C, and even more preferably 2 to 8°C.

[0054] In the present invention, the dialysis treatment time is preferably 2 to 3 hours, more preferably 2.1 to 2.9 hours, and even more preferably 2.2 to 2.8 hours.

[0055] In the present invention, the dialysis treatment can remove the excess impurities in the eluent and improve the purity of prothrombin.

[0056] In the present invention, the dialysate in step (4) needs to be firstly subjected to ultrafiltration desalination treatment, and then the desalinated dialysate is activated.

[0057] In the present invention, in the process of ultrafiltration desalination, an ultrafiltration membrane with a molecular weight cutoff of 10 kDa is used, and ultrafiltration desalination is performed in a continuous diafiltration manner.

[0058] In the present invention, the concentration of the calcium chloride solution in step (5) is preferably 0.05 to 0.15 mol / L, more preferably 0.06 to 0.14 mol / L, and more preferably 0.07 to 0.13 mol / L.

[0059] In the present invention, the temperature of the activation treatment in step (5) is preferably 15 to 25°C, more preferably 16 to 24°C, and even more preferably 17 to 23°C.

[0060] In the present invention, the activation treatment time is preferably 1 to 3 hours, more preferably 1.2 to 2.8 hours, and even more preferably 1.5 to 2.5 hours.

[0061] In the present invention, the ultrafiltration concentration time in step (6) is preferably 4 to 6 hours, more preferably 4.2 to 5.8 hours, and even more preferably 4.5 to 5.5 hours.

[0062] Example 1

[0063] 2800ml of porcine plasma was collected and mixed with 400ml of 0.11mol / L trisodium citrate solution, and then allowed to stand at 5°C for 3h, filtered and centrifuged to obtain supernatant a, and the supernatant a was inactivated by the S / D method, that is, a 0.3% volume ratio concentration of tributyl phosphate aqueous solution and a 1% weight ratio concentration of Tween-80 aqueous solution were mixed with supernatant a and stirred for 3h to obtain solution b. 1000ml of solution b was taken, and solution b was subjected to column chromatography using a DEAE-cellulose anion exchange column, and then eluted with a pH of 7, 0.1mol / L trisodium citrate solution, and the elution flow rate was controlled to be 1mL / min, and then an eluate c was obtained.

[0064] The eluate c is mixed with a 0.02 mol / L aqueous solution of trisodium citrate, dialyzed at 5°C for 2 hours, and then the pH is adjusted to 6.2 to obtain a dialysate d; the dialysate d is ultrafiltered and desalted using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa and a continuous diafiltration method, and then the desalted dialysate d and 70 ml of a 1 mol / L calcium chloride solution are activated at 15°C for 2 hours to obtain a thrombin solution; the thrombin solution is ultrafiltered and concentrated using an ultrafiltration concentrator for 5 hours and then freeze-dried to obtain the thrombin.

[0065] Example 2

[0066] 3200 ml of porcine plasma was collected and mixed with 400 ml of 0.125 mol / L potassium oxalate solution, and then allowed to stand at 8°C for 4 hours, filtered and centrifuged to obtain supernatant a, and the supernatant a was inactivated by the S / D method, that is, a 0.3% volume ratio concentration of tributyl phosphate aqueous solution and a 1% weight ratio concentration of Tween-80 aqueous solution were mixed with supernatant a and stirred for 3 hours to obtain solution b. 1500 ml of solution b was taken, and solution b was subjected to column chromatography using a DEAE-cellulose anion exchange column, and then eluted with an amino acid eluent of pH 8 and 0.2 mol / L, and the elution flow rate was controlled to be 1 mL / min, and then an eluent c was obtained.

[0067] The eluate c is mixed with a 0.01 mol / L aqueous solution of trisodium citrate, dialyzed at 10°C for 2 hours, and then the pH is adjusted to 6.0 to obtain a dialysate d; the dialysate d is ultrafiltered and desalted using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa and a continuous diafiltration method, and then the desalted dialysate d and 140 ml of a 1 mol / L calcium chloride solution are activated at 20°C for 3 hours to obtain a thrombin solution; the thrombin solution is ultrafiltered and concentrated using an ultrafiltration concentrator for 5 hours and then freeze-dried to obtain the thrombin.

[0068] Example 3

[0069] 4200 ml of porcine plasma was collected and mixed with 500 ml of 0.11 mol / L trisodium citrate solution, and then allowed to stand at 5°C for 3 h, filtered and centrifuged to obtain supernatant a. The supernatant a was inactivated by the S / D method, that is, an aqueous solution of tributyl phosphate with a volume ratio concentration of 0.3% and an aqueous solution of Tween-80 with a weight ratio concentration of 1% were mixed with the supernatant a and stirred for 3 h to obtain solution b. 1000 ml of solution b was taken, and solution b was subjected to column chromatography using a DEAE-cellulose anion exchange column, and then eluted with a 0.1 mol / L trisodium citrate solution with a pH of 7, and the elution flow rate was controlled to be 2 mL / min, and then an eluate c was obtained.

[0070] The eluate c is mixed with a 0.02 mol / L aqueous solution of trisodium citrate, dialyzed at 5°C for 2 hours, and then the pH is adjusted to 6.5 to obtain a dialysate d; the dialysate d is ultrafiltered and desalted using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa and a continuous diafiltration method, and then the desalted dialysate d and 75 ml of a 1 mol / L calcium chloride solution are activated at 15°C for 2 hours to obtain a thrombin solution; the thrombin solution is ultrafiltered and concentrated using an ultrafiltration concentrator for 5 hours and then freeze-dried to obtain the thrombin.

[0071] It can be seen from the above embodiments that the present invention provides a method for preparing thrombin, comprising the following steps: adding an anticoagulant to pig plasma, filtering and centrifuging after standing to obtain a supernatant a; inactivating the supernatant a by an S / D method, that is, adding tributyl phosphate and Tween-8 to the supernatant a to obtain a solution b; performing column chromatography on the solution b by using a DEAE-cellulose anion exchange column, and then eluting with an eluent to obtain an eluent c; mixing the eluent c with a trisodium citrate aqueous solution for dialysis treatment, and then adjusting the pH to obtain a dialysate d; mixing the dialysate d with a calcium chloride solution for activation to obtain a thrombin solution; and sequentially performing ultrafiltration concentration and freeze drying on the thrombin solution to obtain the thrombin. The preparation method of the present invention can extract more than 50,000 IU of thrombin product per liter of plasma, greatly improving the extraction efficiency, and only requires a few steps such as centrifugation, sample chromatography, ultrafiltration desalting, activation, ultrafiltration concentration and freeze drying, which is easy to realize industrial production, reduces manual operation and errors, abandons the organic solvent purification method, is harmless to personnel and the environment, and the waste discharge meets environmental protection requirements, reducing environmental pollution.

[0072] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing thrombin, characterized in that: The following steps are involved: (1) adding an anticoagulant to porcine plasma, allowing the plasma to stand, and then filtering and centrifuging to obtain a supernatant a; (2) inactivating the virus in the supernatant a by using the S / D method, i.e., adding tributyl phosphate and Tween-8 to the supernatant a to obtain a solution b; (3) performing column chromatography on solution b using a DEAE-cellulose anion exchange column, and then eluting with an eluent to obtain an eluate c; (4) mixing the eluate c and a trisodium citrate aqueous solution for dialysis treatment, and then adjusting the pH to obtain a dialysate d; (5) mixing the dialysate d and the calcium chloride solution for activation to obtain a thrombin solution; (6) The thrombin solution is sequentially subjected to ultrafiltration concentration and freeze-drying to obtain the thrombin.

2. The preparation method according to claim 1, characterized in that: In step (1), the anticoagulant is one of trisodium citrate solution or potassium oxalate solution; The concentration of the anticoagulant is 0.1 to 0.15 mol / L; The volume ratio of the anticoagulant to blood is 1:7 to 1:

9.

3. The preparation method according to claim 1, characterized in that: The standing time in step (1) is 3 to 5 hours; The standing temperature is 5-15°C.

4. The preparation method according to claim 1, characterized in that: In step (3), the eluent is one or both of trisodium citrate solution and amino acid solution; The concentration of the eluent is 0.05-0.25 mol / L; The pH of the eluent is 6-9.

5. The preparation method according to claim 1, characterized in that: The elution flow rate in step (3) is 1 to 2 mL / min.

6. The preparation method according to claim 1, characterized in that: The concentration of the trisodium citrate solution in step (4) is 0.01 to 0.1 mol / L; The pH is 6.0 to 6.

5.

7. The preparation method according to claim 1, characterized in that: The temperature of the dialysis treatment in step (4) is 0 to 10°C; The dialysis treatment time is 2 to 3 hours.

8. The preparation method according to claim 1, characterized in that: The concentration of the calcium chloride solution in step (5) is 0.05-0.15 mol / L.

9. The preparation method according to claim 1, characterized in that: The temperature of the activation treatment in step (5) is 15 to 25°C; The activation treatment time is 1 to 3 hours.

10. The preparation method according to claim 1, characterized in that: The ultrafiltration concentration time in step (6) is 4 to 6 hours.