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Preparation method of blood coagulation factor XIII

A blood coagulation factor and blood plasma technology, which is applied in the field of medical bioengineering, can solve the problems of increasing the content of toxins such as pyrogens, the reproduction of bacteria and other microorganisms, and the long time of tertiary dissolution, so as to reduce the growth of bacteria, shorten the dissolution time and save time. Effect

Active Publication Date: 2016-04-27
FUJIAN HUAGENE BIOSCI CO LTD
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  • Abstract
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AI Technical Summary

Problems solved by technology

[0006] The three times of dissolution in the production process are too long, which will easily lead to the reproduction of bacteria and other microorganisms, thereby increasing the content of pyrogens and other toxins in the product; too long dissolution time will also easily cause loss of activity, while prolonging the production cycle and increasing costs; The product made by the above process has low purity and low activity

Method used

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  • Preparation method of blood coagulation factor XIII
  • Preparation method of blood coagulation factor XIII
  • Preparation method of blood coagulation factor XIII

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preparation example Construction

[0024] Please refer to figure 1 , the preparation method of blood coagulation factor XIII, comprises the steps:

[0025] Step 1. After adding anticoagulant to the blood, centrifuge and take the supernatant anticoagulant plasma;

[0026] Step 2, adding tributyl phosphate and Tween-80 as virus inactivating agents to supernatant anticoagulated plasma, and adding auxiliary agents to carry out S / D virus inactivation to obtain inactivated plasma;

[0027] Step 3, centrifuging the inactivated plasma obtained in step 2, and taking the supernatant;

[0028] Step 4. Pre-cool the clear liquid obtained in step 3 to 0-4°C, add a saturated ammonium sulfate solution at 0-4°C, and take the precipitate;

[0029] Step 5. Wash the precipitate obtained in step 4 with a saturated ammonium sulfate solution at 0-4°C, break the washed precipitate, add a primary dissolving agent at 0-4°C and stir, and take the clear liquid. The primary dissolving agent is chlorine Potassium chloride solution;

[0...

Embodiment 1

[0052] Please refer to figure 1 , All operations of the process are carried out in a sterile ten thousand-level purification area, among which sub-package and freeze-drying operations are all carried out in a local one-hundred-level purification area, and the operations performed are in line with the operating requirements of the clean area. The production specification is 1.5ml.

[0053] (1) Get 10000ml of aseptically collected pig blood;

[0054] (2) Centrifuge and take 4000ml of supernatant anticoagulated plasma;

[0055] (3) Add 0.3% w / v tributyl phosphate and 1% w / v Tween-80 as virus inactivator to the supernatant anticoagulated plasma of step (2) gained, add the glucose of 0.2mol / L as auxiliary agent, virus inactivation was carried out at 25°C for 6 hours;

[0056] Centrifuge the inactivated plasma, discard the precipitate, and take the supernatant;

[0057] (4) Primary sedimentation: Precool the clear liquid obtained in step (3) to 0-4°C, add 1000ml of saturated amm...

Embodiment 2

[0077] Please refer to figure 1 , All operations of the process are carried out in a sterile ten thousand-level purification area, among which sub-package and freeze-drying operations are all carried out in a local one-hundred-level purification area, and the operations performed are in line with the operating requirements of the clean area. The production specification is 1.5ml.

[0078] (1) Get 24000ml of aseptically collected pig blood;

[0079] (2) Centrifuge and take 12000ml of supernatant anticoagulated plasma;

[0080] (3) Add 0.3% w / v tributyl phosphate and 1% w / v Tween-80 as virus inactivator to the supernatant anticoagulated plasma of step (2) gained, add the glucose of 0.2mol / L as auxiliary agent, virus inactivation was carried out at 25°C for 6 hours;

[0081] Centrifuge the inactivated plasma, discard the precipitate, and take the supernatant;

[0082] (4) Primary sedimentation: precool the clear liquid obtained in step (3) to 0-4°C, add 3000ml of saturated am...

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Abstract

The invention provides a preparation method of a blood coagulation factor XIII. The preparation method has the advantages of high yield, high purity and short production period, and comprises the steps of blood plasma and blood cell separation, virus inactivation, ammonium sulfate graded sedimentation, DEAE-FF chromatographic column treatment, split charging and freeze drying, wherein an auxiliary agent is added during the virus inactivation; the graded sedimentation and dissolution process of the blood coagulation factor XIII is performed in an environment being 0 to 4 DEG C; different dissolving agents are added for performing graded dissolution on precipitates; the dissolution efficiency is improved; the production period is shortened; the bacteria breeding is reduced; and the yield and the purity of the blood coagulation factor XIII are improved. The preparation method of the blood coagulation factor XIII provided by the invention has the beneficial effects that the extraction efficiency is high; more than 10mg of the blood coagulation factor XIII can be extracted from per liter of blood plasma; in a prepared blood coagulation factor XIII freeze-drying product, the purity of the blood coagulation factor XIII is higher than 90 percent; the operation is simple; the production period is short; and the preparation of the blood coagulation factor XIII can be completed in two days.

Description

technical field [0001] The invention belongs to the field of medical bioengineering, and in particular relates to a method for preparing medical blood coagulation factor XIII by using human blood or mammalian blood as a raw material. Background technique [0002] Coagulation factor XIII (also known as fibrin stabilizing factor, fibrin ligase, transglutaminase) is based on the zymogen (A 2 B 2 Tetramer, M r ≈320KD) is a glycoprotein that exists in plasma. It is a plasma protease necessary for normal blood coagulation. After being activated, it acts as a transaminase and can catalyze the cross-linking reaction between soluble fibrin monomer (sFM) molecules. Form γ-chain dimers and large-molecular-weight α-chain polymers to increase the strength of blood clots and accelerate wound healing. [0003] At present, the preparation of coagulation factor XIII at home and abroad is mainly extracted from human plasma and yeast cells, as well as from platelets and embryos. When it is ...

Claims

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Application Information

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IPC IPC(8): C12N9/10
CPCC12N9/1044C12Y203/02013
Inventor 赵晶黄发灿郑登忠章丽丽
Owner FUJIAN HUAGENE BIOSCI CO LTD
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