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Freeze-dried human prothrombin complex and preparation method thereof

A technology of human prothrombin and complexes, which is applied in the field of medicine and biology, can solve the problems of high chromatographic purification costs, unbalanced ratio, and low FVII factor, and achieve the effects of reducing the risk of thrombosis, balancing the ratio, and improving product quality

Pending Publication Date: 2021-11-02
HUALAN BIOLOGICAL ENG INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are two problems in the crude product of human prothrombin complex produced in this way: the titer of FVII and the specific activity of FIX cannot reach a higher level at the same time; the contents of FII and FX are relatively high, about 130% to 160% of FIX
However, the content of FII and FX factors in this process is relatively high, and the proportion of the four factors is not balanced.
[0007] Chinese patent CN201710118663.7 discloses a preparation method of human prothrombin complex, using DEAEA-50 gel to directly adsorb human prothrombin complex from plasma, and packing the adsorbed A-50 gel into a fixed bed In the chromatography column, the peristaltic pump is used to pump the solution for online washing and elution, and the eluate is inactivated by S / D for secondary chromatography purification on a fixed bed, which reduces the loss of the gel by more than 20% during the production process, but The resulting clotting factor has a lower specific activity
[0008] Chinese patent CN201810055362.9 uses DEAE Sephadex A-50 gel for chromatographic purification for the first chromatographic purification, and uses Capto DEAE gel for chromatographic purification for the second chromatographic purification. The activity is higher, but the specific activity of FVII factor is lower
And the cost of chromatographic purification using Capto DEAE gel is relatively high

Method used

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  • Freeze-dried human prothrombin complex and preparation method thereof
  • Freeze-dried human prothrombin complex and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] (1) Centrifugation: after thawing and mixing the fresh frozen healthy human plasma, cryoprecipitate centrifugation was carried out at 0-4°C, and 15 L of the obtained supernatant was collected. Use 0.01mol / L acetic acid solution to adjust the pH value to 7.0±0.2.

[0067] (2) The first gel adsorption: Equilibrate 37.5g of DEAE Sephadex A-50 gel to pH = 7.0±0.2 using equilibrium solution I, mix with the supernatant, stir and adsorb at 5±3°C for 50 minutes, and then filter. Equilibrium solution Ⅰ is 0.01mol / L sodium citrate+0.1mol / L sodium chloride, pH=7.0±0.1.

[0068] (3) Washing: Wash the gel 5 times with 670ml balance solution II (the volume ratio of balance solution II to the gel after the first adsorption can be 1:1, and the volume of 37.5g DEAE Sephadex A-50 gel after swelling about 670ml). Use 670ml washing solution to wash the gel 6 times; balance solution II is 0.01mol / L sodium citrate+0.09mol / L sodium chloride, pH=7.0±0.1; washing solution is 0.01mol / L sodium ...

Embodiment 2～5

[0080] Examples 2-5 are the same as the method steps and conditions in Example 1 of the present invention, the difference is the adsorption conditions for the first adsorption, as shown in Table 1.

[0081] The adsorption condition of the gel adsorption for the first time of table 1 embodiment 1~5

[0082] Gel addition (g / L plasma supernatant) Supernatant pH Example 1 2.5 7.0 Example 2 1.5 6.4 Example 3 2 7.4 Example 4 2.5 6.4 Example 5 1.5 7.4

Embodiment 6～10

[0084] Examples 6-10 are the same as the method steps and conditions in Example 1 of the present invention, and the difference is the washing conditions, as shown in Table 2.

[0085] The washing condition of table 2, embodiment 6~10

[0086] Sodium citrate (mol / L) Sodium chloride (mol / L) Example 6 0.01 0.12 Example 7 0.01 0.15 Example 8 0.01 0.16 Example 9 0.01 0.18 Example 10 0.01 0.20

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Abstract

The invention discloses a freeze-dried human prothrombin complex and a preparation method thereof. The preparation method comprises the following steps: sequentially performing centrifugation, first-time gel adsorption, washing, elution, ultrafiltration, dialysis, S / D inactivation, second-time gel adsorption, second-time washing, second-time elution, second-time ultrafiltration and dialysis on fresh frozen healthy human plasma, and then subpackaging and freeze-drying to obtain the freeze-dried human prothrombin complex. According to the method disclosed by the invention, DEAE Sephadex A-50 gel with relatively low cost is used for purification; through the process of two purification steps, the effects of controlling the F VII factor content, effectively improving the specific activity of FIX and adjusting the proportion of the four factors are achieved respectively.

Description

technical field [0001] The invention relates to a freeze-dried human prothrombin complex and a preparation method thereof, belonging to the field of medical biotechnology. Background technique [0002] The human prothrombin complex contains four coagulation factors II, VII, IX, and X that are synthesized in the liver dependent on vitamin K. Vitamin K deficiency or severe liver disease can lead to deficiencies in these four factors, which in turn lead to coagulation disorders. Infusion of human prothrombin complex increases the concentration of clotting factors in the blood. Therefore, it is mainly used for the treatment of congenital and acquired coagulation factor II, VII, IX, and X deficiency. In addition, it has also been affirmed in the treatment of bleeding symptoms caused by FⅧ inhibitors in hemophilia A patients who have been infused with FⅧ for a long time. In foreign countries, human prothrombin complex is also widely used in the treatment of bleeding symptoms ca...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/64C12N9/74
CPCC12N9/6429C12N9/6437C12N9/644C12N9/6432
Inventor 马小伟赵良超张学成杨保平宋博赵东生
Owner HUALAN BIOLOGICAL ENG INC