Purification method of human fibrinogen

A fibrinogen and buffer technology, applied in the field of purification of human fibrinogen, can solve the problems of complicated purification steps and residual virus inactivator tributyl phosphate

Active Publication Date: 2021-11-26
CHENGDU RONGSHENG PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the purification steps of this method are relatively complicated, and it needs to go through lysine affinity column chromatography and cation exchange column chromatography successively; moreover, the virus inactivation process of this method adopts the S / D method, but the human purified by this method Fibrinogen also contains virus inactivator tributyl phosphate residue

Method used

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  • Purification method of human fibrinogen
  • Purification method of human fibrinogen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1. Purification method of human fibrinogen

[0039] Step 1: Preliminary Purification

[0040] 1. Take the cryoprecipitate prepared from fresh frozen plasma, and dissolve the cryoprecipitate in 20mM Tris buffer solution with a pH of 6.8 at a weight ratio of 1:3 at room temperature to obtain a cryoprecipitate solution;

[0041] 2. Add aluminum hydroxide (the amount of aluminum hydroxide is 2wt.% of the cryoprecipitate) to the cryoprecipitate solution, and stir at room temperature for 15 minutes to carry out adsorption treatment;

[0042] 3. Centrifuge the system obtained after adsorption at 4000g for 20 minutes, take the supernatant and pass it through a 0.45μm filter membrane, and keep the filtrate;

[0043] Step 2: Virus inactivation

[0044] Carry out virus inactivation to gained filtrate by S / D method: get the first step gained filtrate, add polysorbate 80 (polysorbate 80 consumption is the 1wt.% of filtrate) and tributyl phosphate (tributyl phosphate consu...

experiment example 1

[0050] Experimental example 1. Adsorption effect evaluation

[0051] 1. Experimental method

[0052] The cryoprecipitate in the first step of Example 1 and the filtrate after the adsorption treatment were respectively used as test samples to test the purity of human fibrinogen and the adsorption effects of coagulation factors II, VII, IX, and X in the samples before and after preliminary purification.

[0053] 2. Experimental results

[0054] Table 1 Human fibrinogen and adsorbed coagulation factors II, VII, IX, X (n=3) in samples before and after preliminary purification

[0055] Test indicators Before initial purification After initial purification Protein content mg / ml (n=8) 34.65 33.01 Coagulable protein content mg / ml (n=8) 21.88 21.49 Purity % (n=8) 63.15 65.10 FII IU / ml (n=2) 0.32 0.11 FVII IU / ml (n=2) 0.54 0.08 FIX IU / ml (n=2) 0.85 0.23 FX IU / ml (n=2) 0.13 0.04

[0056] In Table 1, the protein content ...

experiment example 2

[0058] Experimental example 2. Evaluation of virus inactivation effect

[0059] 1. Experimental method

[0060] The sample to be loaded on the column obtained after the virus inactivation in the second step of Example 1 was used as the test sample to evaluate the virus inactivation effect.

[0061] 2. Experimental results

[0062] Table 2. SD virus inactivation effect (n=3)

[0063] indicator virus PRV virus Sindbis virus HIV virus Inactivation effect ≥4.75logTCID 50 / ml

[0064] As can be seen from the results in Table 2, carrying out SD virus inactivation with the method of the present invention can effectively inactivate lipid-enveloped viruses and reach the national standard.

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Abstract

The invention discloses a purification method of human fibrinogen and belongs to the field of plasma product preparation processes. The method comprises the following step: carrying out ion exchange chromatography on fibrinogen subjected to virus inactivation, thereby obtaining purified fibrinogen, wherein a buffer solution adopted by the ion exchange chromatography comprises an equilibrium buffer solution, a washing buffer solution and an eluting buffer solution. Experimental results show that by means of the purification method disclosed by the invention, the content and purity of the human fibrinogen in an obtained product can be obviously improved, and virus inactivator residues in the product can be obviously reduced. The method disclosed by the invention is simple and convenient to operate and low in cost and has a good industrial application prospect.

Description

technical field [0001] The invention belongs to the field of plasma product preparation technology, in particular to a method for purifying human fibrinogen. Background technique [0002] Human fibrinogen (Fibrinogen, Fg), also known as human coagulation factor Ⅰ, is mainly synthesized by liver parenchymal cells and is one of the main components of plasma proteins. The content of fibrin in plasma is rich, and the normal human plasma is about 2-4g / L, is one of the "central" proteins in the coagulation system. [0003] Human fibrinogen is the final substrate of the successive activation of coagulation factors in the coagulation process, and has the function of hemostasis. In addition to directly participating in the late stage of the coagulation process, it can also mediate platelet aggregation and affect blood viscosity. In the common pathway of blood coagulation, thrombin first cleaves Arg16-Gly17 at the amino terminals of the two Aα chains of fibrinogen to release a pair ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/75C07K1/18
CPCC07K14/75
Inventor 鲁涛牟蕾李伟余伟王黔川
Owner CHENGDU RONGSHENG PHARMA
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