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Influenza virus subunit vaccine purification method and applications thereof

A technology of subunit vaccine and influenza virus, applied in the field of preparation method and vaccine prepared by the method

Inactive Publication Date: 2018-06-15
AB&B BIO TECH CO LTD JS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, impurities such as ovalbumin in the vaccine cannot be completely removed. Therefore, it is necessary to continuously improve the process during the research and development process, reduce the content of impurities as much as possible, increase the content of effective antigenic substances, and continuously improve the safety and effectiveness of the product.

Method used

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  • Influenza virus subunit vaccine purification method and applications thereof
  • Influenza virus subunit vaccine purification method and applications thereof
  • Influenza virus subunit vaccine purification method and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] (1) Proliferation and concentration of virus

[0063] After diluting the working seed lot virus to a final concentration of 2.0lgEID50 / ml, inoculate the allantoic cavity of chicken embryos at a dose of 0.2ml / embryo, culture at 33°C for 48 hours, screen live chicken embryos, and place cold embryos at 4°C for 12 hours. Hours later, the allantoic fluid was harvested, and the allantoic fluid harvested from the single-type influenza virus was centrifuged and clarified and combined into a monovalent virus pooled liquid, and the monovalent virus pooled liquid was concentrated by ultrafiltration with a molecular weight cut-off ultrafiltration membrane of 1 million, and the concentration factor was 20 times .

[0064] (2) Purification before virus lysis

[0065] Prepare 15%, 30% and 60% sucrose solutions respectively, put the rotor into the ultracentrifuge, start and install the rotor, close the hatch, and set the speed at 1000rpm at the same time, after the rotor is stable, st...

Embodiment 2

[0075] A quadrivalent influenza virus subunit vaccine, each dose of the quadrivalent influenza virus subunit vaccine contains two lineage BY and BV strains of H1N1 type, H3N2 type and B type, and the content of each valent antigen is 30-36 μg / mL.

[0076] (1) Proliferation and concentration of virus

[0077] After diluting the batch of working seeds to a final concentration of 5.0lgEID50 / ml, inoculate the allantoic cavity of chicken embryos at a dose of 0.2ml / embryo, culture at 35°C for 72 hours, screen live chicken embryos, and place them in a cold place at 8°C. After 24 hours of embryos, the allantoic fluid was harvested, and the allantoic fluid harvested from the monotype influenza virus was centrifuged and clarified and combined into a monovalent virus pooled fluid. Use a 1 million molecular weight cut-off ultrafiltration membrane to conduct ultrafiltration and concentration of the monovalent virus pool solution, and the concentration factor is 60 times.

[0078] (2) Pu...

Embodiment 3

[0090] The influenza virus cracking electron micrograph of embodiment 3 different purification processes

[0091] 1. Materials:

[0092] H1N1 Influenza Virus Concentrate

[0093] 2. Method:

[0094] The concentrated solution of H1N1 influenza virus was lysed by two different virus purification processes.

[0095] The first is the pre-cleavage purification plus separate cleavage process adopted by the existing three-step purification method: first, the virus concentrate is subjected to sucrose density gradient centrifugation, and the absorbance value at 280nm is recorded with an ultraviolet spectrophotometer to detect the hemagglutination drops in each section degree, collect the target peak with high hemagglutination titer as the virus purification solution, and then add Triton N-101 lysing agent to the virus purification solution according to the final concentration of 1.0%. Samples were taken for electron microscopy examination.

[0096] The second is the pre-lysis purif...

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Abstract

The invention provides an influenza virus subunit vaccine purification method, which comprises a four-step purification technology, and a vaccine prepared thereby. The provided influenza virus subunitvaccine four-step purification technology overcomes the shortages of a conventional three-step purification technology; reduces the content of ovalbumin in the vaccine, enhances the virus splitting effect, and increases the contents of effective antigen substances such as HA.

Description

technical field [0001] The invention belongs to the field of medicine, and relates to a method for preparing an influenza vaccine and a vaccine prepared by the method. Background technique [0002] Influenza virus (influenza virus, referred to as influenza virus) is an acute respiratory infectious disease that causes zoonosis in humans, poultry and animals. According to the structure and gene characteristics of hemagglutinin and neuraminidase on the surface of the virus, it is divided into many subtypes. Currently, there are 16 subtypes (H1-H16) of hemagglutinin and 10 subtypes of neuraminidase. Type (N1 ~ N10). [0003] Getting a flu vaccine is the best way to prevent the flu from happening and spreading. Influenza virus subunit vaccine is obtained by cracking the virus with a lysing agent. The vaccine contains hemagglutinin / neuraminidase surface antigen and other antigens. The virus envelope is destroyed and the lipid is removed, which has a high immunogenicity Sex, les...

Claims

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Application Information

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IPC IPC(8): A61K39/145A61P31/16
CPCA61K39/12A61K2039/5252C12N2760/16134
Inventor 安有才徐奇张玉辉于海龙陈辉黎冷文娜张宏栋张焕陈晓芬张慧萍刘鹏安蕊刘冰冰
Owner AB&B BIO TECH CO LTD JS
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