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Lyophilized inactivated Japanese encephalitis vaccine

A Japanese encephalitis and inactivation technology, applied in the field of new freeze-dried inactivated Japanese encephalitis whole virus vaccine, can solve the problems of unstable purified vaccine and low content of impurity protein, and achieve easy quantitative and qualitative, clear composition, high quality Easy-to-control effects

Active Publication Date: 2014-04-02
天津嘉诚顺隆商贸有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, due to the low content of impurity proteins in the purified vaccine, the total protein content combined with the effective protein of the vaccine is only within 100 micrograms, and the virus particles are completely exposed, so the purified vaccine is more unstable than the unpurified vaccine

Method used

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  • Lyophilized inactivated Japanese encephalitis vaccine
  • Lyophilized inactivated Japanese encephalitis vaccine
  • Lyophilized inactivated Japanese encephalitis vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Comparison of freeze-dried state of different formulation excipients

[0039] a) Preparation of Japanese encephalitis virus inactivation stock solution

[0040] Use a 175cm2 culture flask (Corning Company) to resuscitate a 136-generation working cell bank Vero cell (ATCC, CCL81), culture it in a carbon dioxide incubator at 37 degrees for 3-5 days, discard the culture medium, and add 0.25% trypsin (GIBCO) Digest for 3-5 minutes, add fresh growth medium (serum-free medium PV2, Tianjin Bairuoke Company) to disperse the cells by blowing, 1:6 split into six 175cm2 culture flasks, and culture under the same conditions. In this way, 24 10-layer 1750 flasks (Corning Company) were amplified, which were digested and inoculated into a bioreactor after forming a dense monolayer.

[0041] Adopt the 7.5L cell culture reactor produced by APPLIKAN; cytodex1 microcarrier (GE Company), the concentration is 20g / l. Serum-free medium PV2 was used, the culture temperature was 37 degrees, t...

Embodiment 2

[0074] Comparison of Stability of JE Vaccines with Different Concentrations of Trehalose

[0075] a) Preparation of JE vaccine inactivated stock solution

[0076] The method was the same as that in Experiment 1, and the antigen concentration of the inactivated stock solution was measured to be 30 EU / ml by the double-antibody sandwich method.

[0077] b) On the basis of Experiment 1 formula B, add different concentrations of trehalose (2%, 0.5%, 0%), then place the vaccine at 4 degrees and 37 degrees respectively, take samples regularly, and use the double antibody sandwich method to determine the The antigenic content of the vaccine, and the vaccine potency were determined using a mouse immune neutralizing antibody assay.

[0078] c) Antigen results at 37 degrees are as follows: figure 1 , the antigen was measured every two days. With the prolongation of the storage time of the vaccine at 37 degrees, the antigen content decreased, but the decline rate of the vaccine containi...

Embodiment 3

[0082] Stability test results of freeze-dried vaccine

[0083] a) Vaccine preparation

[0084] Vero cells, passage 139, recovered and expanded to a 5L reactor; cytodex1 microcarrier, 10g / L;

[0085] Japanese encephalitis virus: the Vero cell passage virus species P3V5 of the P3 strain. MOI, 0.02

[0086] Cell culture temperature: 36.5 degrees, stirring speed: 80RPM, 80% dissolved oxygen, pH7.2;

[0087] Virus culture temperature: 34.5 degrees, stirring speed: 80rpm, 80% dissolved oxygen, pH7.8

[0088]The virus harvested solution was filtered at 1.0+0.22 microns, concentrated 100 times with a 100KD ultrafiltration membrane, centrifuged through a sucrose density gradient, desugared by ultrafiltration, inactivated with formaldehyde, and the antigen concentration of the inactivated stock solution was determined.

[0089] b) Preparation of semi-finished products:

[0090] Prepare the semi-finished product with 0.01M phosphate buffer containing maltose, lactose, arginine and t...

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Abstract

The invention provides a lyophilized inactivated Japanese encephalitis vaccine, comprising: (a) inactivated Japanese encephalitis totivirus; (b) stabilizer, which comprises maltose, lactose, and optionally mycose, mannite, sorbitol and / or amino acid; and optionally (c) buffer, surfactant, isotonic regulator and / or chelating agent.

Description

technical field [0001] The invention relates to a novel freeze-dried and inactivated Japanese encephalitis whole virus vaccine. Background technique [0002] The pathogen of epidemic encephalitis B (JE) was discovered in Japan in 1934, so it is also called Japanese Encephalitis. Japanese encephalitis virus belongs to the flavivirus genus of Togaviridae. The spherical virus particles have capsules outside, and are sensitive to temperature, ether and other organic solvents. The Japanese encephalitis virus is transmitted by mosquitoes and is prevalent in summer and autumn. There are about 50,000 cases of the disease every year in the world, and the main endemic areas are countries such as southern my country, Southeast Asia, and tropical regions. WHO recommends that all JE-endemic countries should implement JE vaccine immunization. [0003] JE vaccines currently used internationally are mainly inactivated whole virus JE vaccines and live attenuated JE vaccines. Maintaining g...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/12A61K47/26A61K9/19A61P31/14
CPCY02A50/30
Inventor 石慧颖任秀宝
Owner 天津嘉诚顺隆商贸有限公司
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