Method for carrying out virus inactivation on pig fibrinogens through freeze-drying and heating

A technology of dry heat virus inactivation and fibrinogen, which is applied in the field of separation and purification of animal blood products, can solve the problems of medical pig blood product virus inactivation, reduce the risk of animal-derived virus infection, reduce requirements, and improve the effect good effect

Active Publication Date: 2013-04-17
浙江赛灵特医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The invention discloses a method for inactivating porcine fibrinogen by freeze-drying and heating viruses, which solves the problem of virus inactivation in the production of medical pig blood products, reduces the risk of animal-derived virus infection, and ensures the efficiency of virus inactivation Effect

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] a. First, add ammonium sulfate to the pig plasma to a concentration of 20% saturation for salting out, stir until the crude fibrinogen is precipitated, and collect the fibrinogen after standing; dilute the fibrinogen and add an appropriate amount of anticoagulant; fibrin The original solution was filtered through filters with a pore size of 5 μm, 0.45 μm, and 0.22 μm, respectively, and ultrafiltration was carried out with a polysulfone ultrafiltration membrane with a relative molecular weight cut-off of 80,000 to 100,000; an appropriate amount of tributyl phosphate and polysorbate was added to carry out S / D virus was inactivated and filtered through a 0.45 μm filter; fibrinogen concentrate was obtained after ethanol precipitation;

[0025] b. Adding 1% by mass of the group protecting agent is mannitol, sucrose and glycine;

[0026] c. pH adjustment to 7.8;

[0027] d. Sterile filtration, through a filter with a pore size of 0.2 μm (20 inches of polyvinylidene fluoride)...

Embodiment 2

[0032] a. First, add ammonium sulfate to the pig plasma to a concentration of 20% saturation for salting out, stir until the crude fibrinogen is precipitated, and collect the fibrinogen after standing; dilute the fibrinogen and add an appropriate amount of anticoagulant; fibrin The original solution was filtered through filters with a pore size of 5 μm, 0.45 μm, and 0.22 μm, respectively, and ultrafiltration was carried out with a polysulfone ultrafiltration membrane with a relative molecular weight cut-off of 80,000 to 100,000; an appropriate amount of tributyl phosphate and polysorbate was added to carry out S / D virus was inactivated and filtered through a 0.45 μm filter; fibrinogen concentrate was obtained after ethanol precipitation;

[0033] b adding 1% by mass of the group protecting agent is mannitol, sucrose and glycine;

[0034] c adjust the pH to 8.5;

[0035] d. Sterile filtration, through a filter with a pore size of 0.2 μm (20 inches of polyvinylidene fluoride); ...

Embodiment 3

[0041] a. After the pig plasma collection is completed, operations such as salting out, multi-step filtration, S / D virus inactivation and ultrafiltration are performed to obtain fibrinogen concentrate;

[0042] b. Add a group protecting agent, the group protecting agent can adopt mannitol, sucrose and glycine;

[0043] c. pH adjusted to 8.2;

[0044] d. Sterile filtration, through a filter with a pore size of 0.2 μm (20 inches of polyvinylidene fluoride);

[0045] e. Using 2.5mL aseptic filling, freeze-drying and capping operation, to obtain the freeze-dried product of porcine fibrinogen;

[0046] f. The porcine fibrinogen freeze-dried product was kept in a water-proof incubator at a controlled temperature of 63°C for 139 hours to inactivate the virus by dry heat.

[0047] g. The Sindbis virus titer was 1.0LgPFU / mL and the PPV virus titer was 0.49LgTCID detected by the 6-well plaque method 50 / 0.1ml.

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Abstract

The invention discloses a method for carrying out virus inactivation on pig fibrinogens through freeze-drying and heating. The method comprises the following steps: carrying out pretreatment on pig plasmas so as to obtain a concentrated pig fibrinogen solution; adding a group protectant into the concentrated solution; adjusting the pH value of the obtained mixture to 7.5-8.5; enabling the obtained mixture to pass a filter with a bore diameter of 0.2mu m; carrying out aseptic filling, freeze-drying and capping seal on the obtained solution by using 2.5mL perfume bottle so as to obtain a freeze-dried pig fibrinogen product; and carrying out heat preservation on the freeze-dried pig fibrinogen product 139-141 hours at a temperature of 63-67 DEG C so as to carry out hot air viral inactivation. The method disclosed by the invention is verified by National Institute for the Control of Pharmaceutical and Biological Products (No.624 [2008] of NICPBP), through adding Sindbis viruses and PPV viruses to carry out verification, the inactivation effect is remarkable. In the method, through using a simple and feasible freeze-drying product virus inactivating method, the requirements for drying and sterilizing aseptic environments are reduced, the risk of animal-based virus infection in pig fiber products is reduced, and the influence (arising from the traditional virus inactivation) on the bioactivities such as proteins and the like of a product is avoided.

Description

technical field [0001] The invention belongs to the technical field of separation and purification of animal blood products, and in particular relates to a virus inactivation control process in the production process of medical biological products. Background technique [0002] Virus inactivation or removal technologies for traditional blood products mainly include: [0003] Pasteur heat treatment method: The theoretical basis of this method is to make the destruction rate of the virus structure far greater than that of the protein structure through the selection of temperature and action time. The liquid is heated at 60°C, treated for 20 hours, and in the presence of protective coagulation factor stabilizers, it can effectively inactivate various viruses in blood products. [0004] S / D treatment method: The principle is that organic solvents can cause lipids to fall off from the surface of the virus, destroying the structure of the virus and losing its infectious activity....

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K38/36A61K9/19C07K14/75
Inventor 汤瑜黄云战刘金明
Owner 浙江赛灵特医药科技有限公司
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