Method for removing endotoxin in influenza virus stock solution

A technology of influenza virus and endotoxin, which is applied in the direction of viral peptides, antiviral agents, peptide sources, etc., can solve the problems of expensive carrier, low processing capacity, and short carrier life, and achieve constant hemagglutination titer and removal rate High, optimized quality results

Active Publication Date: 2009-08-12
INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In addition, affinity chromatography systems are used to selectively remove endotoxins. In terms of carrier costs, the carriers in these systems are expensive and difficult to regenerate, resulting in short life of the carrier and the problem of small capacity in the chromatographic system.
U.S. Patent (US4885168) adopts low-molecular-weight chitosan, and utilizes positively charged amino groups on chitosan to interact with negatively charged nucleic acid and endotoxin to achieve the purpose of removing nucleic acid and endotoxin, but the utilization rate of chitosan Low
D.Petsch, T.C.Beeskow et al. (1997, Journal of Chromatography B.693, 79-91) used sodium deoxycholate or hexamethylenediamine as the affinity ligand, and used the microporous nylon membrane as the medium to make an affinity Membrane; a single affinity membrane can remove endotoxin in buffer or bovine serum albumin; but the flow rate is low, the resistance is high, the

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] A, in the centrifuge tube that the stock solution of influenza virus of 50ml is housed, add the polyethylene glycol monooctylphenol ether surfactant Triton X-114 medium 0.5ml that final concentration is 1%, mix;

[0022] B. Incubate in a water bath at a temperature of 37±0.5°C for 10 minutes, so that the polyethylene glycol monooctylphenol ether surfactant Triton X-114 medium and the influenza virus stock solution form a phase, and the solution is clearly separated after 30 minutes after taking out;

[0023] C. Centrifuge at room temperature and 15000rpm for 8 minutes, absorb the supernatant and transfer to another centrifuge tube;

[0024] D. In the centrifuge tube of step C, add polyethylene glycol monooctylphenol ether surfactant Triton X-114 medium with a final concentration of 1%, and repeat steps A-C for 3 times, and collect the resulting supernatant , which is the stock solution of influenza virus from which endotoxin has been removed.

[0025] Adopt convention...

Embodiment 2

[0027] A. Add 1ml of polyethylene glycol monooctylphenol ether surfactant Triton X-114 medium with a final concentration of 2% in a centrifuge tube containing 50ml of influenza virus stock solution, and mix well;

[0028] B. Incubate in a water bath at a temperature of 37±0.5°C for 5 minutes, so that the polyethylene glycol monooctylphenol ether surfactant Triton X-114 medium and the influenza virus stock solution form a phase, and the solution is clearly separated in 20 minutes after taking out;

[0029] C. Centrifuge at room temperature and 12000 rpm for 3 minutes, absorb the supernatant and transfer to another centrifuge tube;

[0030] D. In the centrifuge tube of step C, add polyethylene glycol monooctylphenol ether surfactant Triton X-114 medium with a final concentration of 2%, and repeat steps A to C twice, and collect the supernatant , which is the stock solution of influenza virus from which endotoxin has been removed.

[0031] Adopt conventional inspection methods ...

Embodiment 3

[0033] A, in the centrifuge tube that the stock solution of influenza virus of 50ml is housed, add the polyethylene glycol monooctylphenol ether surfactant Triton X-114 medium 1.5ml that final concentration is 3%, mix;

[0034] B. Incubate in a water bath at a temperature of 37±0.5°C for 8 minutes, so that the polyethylene glycol monooctylphenol ether surfactant Triton X-114 medium and the influenza virus stock solution form a phase, and the solution is clearly separated after 25 minutes after taking out;

[0035] C. Centrifuge at room temperature and 10,000 rpm for 5 minutes, absorb the supernatant and transfer to another centrifuge tube;

[0036] D. In the centrifuge tube of step C, add polyethylene glycol monooctylphenol ether surfactant Triton X-114 medium with a final concentration of 3%, and repeat steps A-C for 2 times, and collect the resulting supernatant , which is the stock solution of influenza virus from which endotoxin has been removed.

[0037]Adopt convention...

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PUM

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Abstract

The invention provides a method for removing toxin from flu virus stock solution, which comprises extraction separation and is characterized in that the medium-polyethylene glycol mono-octyl phenolic ether with the final concentration of 0.5-5% (volume percentage concentration) is added into the flu virus stock solution and used for extracting and separating protein and endotoxin in the flu virus stock solution. In the condition of without changing the former natures of the flu virus stock solution, the endotoxin in the flu virus stock solution can be effectively removed to cause that the contents of the endotoxin are reduced to be less than 0.25 EU/Ml and active ingredients in the flu virus stock solution are remained, thus achieving the effect of optimizing quality. The method has the advantages that the operation is simple, the removal rate of endotoxin is high, the recovery rate of the total protein can reach more than 85%, hemagglutination titer is constant, the recovery rate of hemagglutinin content reaches more than 99%, and the obtained flu virus stock solution is safe and reliable.

Description

technical field [0001] The invention relates to a method for removing endotoxin, in particular to a method for removing endotoxin from influenza virus stock solution during the production process of influenza virus stock solution, and belongs to the field of biotechnology. technical background [0002] With the emphasis on the safety of biological products, the control of their pyrogens is becoming more and more stringent. In the production process of biological products, endotoxin pollution is often caused by factors such as raw materials, production environment, and personal operation. Therefore, the production link is a major source of endotoxin production. To prevent endotoxin pollution, it must be Take effective process control and strict preventive measures. [0003] Most of the existing methods destroy or remove endotoxins according to the characteristics of endotoxin molecules. Specifically, it includes using conventional ultrafiltration membrane, ion exchange chro...

Claims

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Application Information

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IPC IPC(8): C07K14/11A61P31/16
Inventor 李映波黄秋香毕湖冰张新文高菁霞蔡玮刘泽姚忠萍姜述德
Owner INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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