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

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

Active Publication Date: 2011-10-12
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

Benefits of technology

This technology allows for easy destruction or removal from harmful substances like bacteria without requiring high temperatures that could damage sensitive materials such as proteins used by animals during production processes. It also prevents these agents from infecting humans with them while still maintaining their beneficial properties when they are applied topically over large areas.

Problems solved by technology

Technics Problem addressed in this patents are how to safely remove unwanted bacteria (virus) without damaging their own healthy cells during production processes like manufacturing plasma albumin concentrates used in pharmaceuticals.

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 precipitates, and collect the fibrinogen after standing; dilute the fibrinogen and add an appropriate amount of anticoagulant; remove the fibrinogen The original solution was filtered through filters with pore diameters of 5μm, 0.45μm, and 0.22μm, and ultrafiltration was performed with a polysulfone ultrafiltration membrane with a relative molecular weight cut-off of 80,000 to 100,000; adding appropriate amounts of tributyl phosphate and polysorbate to perform S / D virus is inactivated and filtered through a 0.45μm filter; the fibrinogen concentrate is obtained after ethanol precipitation;

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

[0026] c. Adjust the pH to 7.8;

[0027] d. Sterilization filtration, through a 0.2μm (20 inch polyvinylidene fluoride) filter;

[0028] e. Use 2.5mL aseptic filling, free...

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 precipitates, and collect the fibrinogen after standing; dilute the fibrinogen and add an appropriate amount of anticoagulant; remove the fibrinogen The original solution was filtered through filters with pore diameters of 5μm, 0.45μm, and 0.22μm, and ultrafiltration was performed with a polysulfone ultrafiltration membrane with a relative molecular weight cut-off of 80,000 to 100,000; adding appropriate amounts of tributyl phosphate and polysorbate to perform S / D virus is inactivated and filtered through a 0.45μm filter; the fibrinogen concentrate is obtained after ethanol precipitation;

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

[0034] c Adjust the pH to 8.5;

[0035] d. Sterilization filtration, through a 0.2μm (20 inch polyvinylidene fluoride) filter;

[0036] e. Add test virus 0.25ml Sindbis vir...

Embodiment 3

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

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

[0043] c. Adjust the pH to 8.2;

[0044] d. Sterilization filtration, through a 0.2μm (20 inch polyvinylidene fluoride) filter;

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

[0046] f. Put the freeze-dried porcine fibrinogen in a water-proof thermostat at a controlled temperature of 63°C for 139 hours to inactivate the dry heat virus.

[0047] g. The titer of Sindbis virus was 1.0LgPFU / mL and the titer of PPV virus was 0.49LgTCID by 6-well disc 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

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Claims

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Application Information

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Owner 浙江赛灵特医药科技有限公司
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