A method for simultaneously separating and purifying blood coagulation factors ix, x and ⅶ from human plasma

A technology for the simultaneous separation of coagulation factors, applied in chemical instruments and methods, coagulation/fibrinolytic factors, peptide preparation methods, etc., can solve the problems of long time-consuming and cumbersome steps, and achieve short time-consuming, simple operation, and realization Take full advantage of the effect

Active Publication Date: 2021-03-30
HUALAN BIOLOGICAL ENG INC +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, it is reported in the literature that the commonly used technical means for the separation and purification of blood coagulation factors II, VII and IX are adsorption, precipitation, ion exchange chromatography or affinity chromatography. One or two of the coagulation factors are separated and purified, and there is no technical scheme or a set of process routes that can simultaneously realize the separation and purification of blood coagulation factors II, VII and IX

Method used

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  • A method for simultaneously separating and purifying blood coagulation factors ix, x and ⅶ from human plasma
  • A method for simultaneously separating and purifying blood coagulation factors ix, x and ⅶ from human plasma
  • A method for simultaneously separating and purifying blood coagulation factors ix, x and ⅶ from human plasma

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Effect test

Embodiment 1

[0036] The technical process of a method for simultaneously separating and purifying blood coagulation factors IX, X and VII from human plasma according to the present invention is as follows: figure 1 , the specific steps include:

[0037] 1. Preparation of prothrombin complex

[0038] S101. Plasma centrifugation to remove cryoprecipitate. Pre-cool the centrifuge in advance. Melt the plasma and centrifuge at 2.0°C to separate and remove the cryoprecipitate. After collection and separation, the plasma is heated to 6.0°C and filtered step by step with 10.0um and 1.0um filter elements until adsorption Can.

[0039] S102. Plasma adsorption and gel washing The adsorption gel medium is DEAE-Sephadex-A50, and the gel balance solution is 10mM citric acid-sodium citrate buffer containing 75mM sodium chloride, pH 7.0. Pump the adsorption equilibrium liquid with 2 times the volume of the gel into the adsorption tank, stir and mix evenly, discard it by suction filtration, and repeat un...

Embodiment 2

[0063] 1. Preparation of prothrombin complex

[0064] S101 Plasma centrifugation to remove cryoprecipitate Pre-cool the centrifuge in advance, melt the plasma and centrifuge at 0°C to separate and remove cryoprecipitate. After collection and separation, the plasma is heated to 4.0°C and filtered to the adsorption tank with a 10μm filter element.

[0065] S102 plasma adsorption and gel washing The adsorption gel medium is DEAE-Sephadex-A50, and the gel balance solution is 15mM citric acid-sodium citrate buffer containing 50mM sodium chloride, pH 6.0. Pump the adsorption balance liquid with 1 times the volume of the gel into the adsorption tank, stir and mix well, discard it by suction filtration, and repeat until the pH value of the suction filtrate is 6.0. After mixing the plasma and the gel in a certain ratio, keep it at 8.0°C, stir and absorb for 20 minutes, and filter the plasma to collect the gel.

[0066] S103 Gel equilibration, washing, and elution The gel washing solut...

Embodiment 3

[0085] 1. Preparation of prothrombin complex

[0086] S101 Plasma centrifugation to remove cryoprecipitate Pre-cool the centrifuge in advance, centrifuge the plasma at 8°C after melting, separate and remove cryoprecipitate, collect and separate and filter to the adsorption tank with a 0.2μm filter element.

[0087] S102 plasma adsorption and gel washing The adsorption gel medium is DEAE-Sephadex-A50, and the gel balance solution is 5mM citric acid-sodium citrate buffer containing 100mM sodium chloride, pH 8.0. Pump 1.5 times the gel volume of the adsorption equilibrium liquid into the adsorption tank, stir and mix well, discard by suction filtration, and repeat until the pH value of the suction filtrate is 8.0. After mixing the plasma and the gel in a certain ratio, stir and absorb for 60 minutes, and collect the gel by suctioning the plasma.

[0088] S103 Gel equilibration, washing, and elution The gel washing solution is 100mM sodium chloride, 5mM citric acid-sodium citrate...

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Abstract

The invention discloses a method for simultaneously separating and purifying coagulation factors IX, X and VII from human plasma, which comprises the following steps: performing centrifugal impurity removal, gel adsorption and ultrafiltration concentration to prepare a prothrombin compound; separating the coagulation factors VII and a mixed solution containing IX and II through an anion exchange resin column; separating the coagulation factor II and IX from the mixed solution containing IX and II by affinity chromatography. According to the method, by combining anion exchange chromatography with heparin affinity chromatography, the separation and preparation of three coagulation factors II, VII and IX at the same time can be achieved; the method has the advantages of high raw material utilization, simple operation and short time consumption; meanwhile, by detecting the electric signals in the chromatography process, the corresponding coagulation factors are accurately collected, the purity of the coagulation factors is effectively improved, and the economic benefit is improved.

Description

technical field [0001] The invention relates to the technical field of separation and purification of biological macromolecules, in particular to a method for simultaneously separating and purifying blood coagulation factors IX, X and VII from human plasma. Background technique [0002] Coagulation factors are various protein components involved in the blood coagulation process. They are activated during bleeding, adhere to platelets and plug wounds on blood vessels. They are specific drugs for the treatment of hemophilia. At present, coagulation factors VII, VIII, IX and prothrombin complexes (mainly coagulation factors II, VII, IX, and X) have been developed into drugs for the treatment of hemophilia A or B. [0003] Coagulation factor IX (FIX for short) is clinically used in the treatment of hemophilia B patients, so further purification of high-purity coagulation factor IX from the prothrombin complex has clinical application value. In addition, blood coagulation factor...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/745C07K14/755C07K1/36C07K1/34C07K1/18
CPCC07K14/745C07K14/755
Inventor 郭敬先马小伟刘晓刚张学成张宝献
Owner HUALAN BIOLOGICAL ENG INC
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