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Method for producing avian influenza vaccine by adopting MDCK cell line and product of avian influenza vaccine

A cell line, avian influenza technology, applied in the field of vaccine preparation, can solve the problems of decreased virus yield, unsuitable for large-scale culture, and unable to meet the needs of virus vaccine production, etc.

Pending Publication Date: 2020-09-29
ZHAOQING DAHUANONG BIOLOGIC PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are some defects in this culture method. For example, the virus yield is reduced due to the limitation of the surface area of ​​the substrate in the adherent culture; Serum culture technology is an effective tool to elucidate the basic issues of cell generation, proliferation, differentiation and gene expression regulation, but even serum-free adherent culture is only suitable for experimental research, not for large-scale culture, and cannot meet the actual production of virus vaccines need

Method used

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  • Method for producing avian influenza vaccine by adopting MDCK cell line and product of avian influenza vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] A kind of method adopting MDCK cell line to produce bird flu vaccine comprises the steps:

[0048] (1) Cell culture: Mix DMEM, F12 and RPMI1640 evenly in a ratio of 3:1:1, use this as the basal medium and add 10% FBS, revive and culture the adherent MDCK cells, and wait until the cells grow to dense cells After layering, digested with 0.25% EDTA-trypsin, passed 5 times; gradually reduced the proportion of DMEM-F12-RPMI1640 medium containing 10% FBS, increased the proportion of serum-free DMEM-F12-RPMI1640 medium, so that the serum content gradually changed from 10 % reduced to 0%;

[0049] After digesting the well-growing MDCK cells adapted to serum-free culture with 0.25% EDTA-trypsin, culture on a shaking table at a speed of 40r / min; after the cell growth rate is stable, subculture, gradually expand the culture, and gradually increase the speed of the shaking table , until acclimatized to a stable proliferation of serum-free full suspension culture MDCK cell line;

...

Embodiment 2

[0058] A kind of method adopting MDCK cell line to produce bird flu vaccine comprises the steps:

[0059] (1) Cell culture: Mix DMEM, F12 and RPMI1640 evenly in a ratio of 3:1:1, use this as the basal medium and add 10% FBS, revive and culture the adherent MDCK cells, and wait until the cells grow to dense cells After layering, digested with 0.25% EDTA-trypsin, passed 5 times; gradually reduced the proportion of DMEM-F12-RPMI1640 medium containing 10% FBS, increased the proportion of serum-free DMEM-F12-RPMI1640 medium, so that the serum content gradually changed from 10 % reduced to 0%;

[0060] After digesting the well-growing MDCK cells adapted to serum-free culture with 0.25% EDTA-trypsin, culture on a shaking table at a speed of 40r / min; after the cell growth rate is stable, subculture, gradually expand the culture, and gradually increase the speed of the shaking table , until acclimatized to a stable proliferation of serum-free full suspension culture MDCK cell line;

...

Embodiment 3

[0069] This embodiment illustrates the inspection and measurement and effect experiment of the vaccine product prepared in Example 2.

[0070] 1. Inspection of semi-finished products:

[0071] Sterility test: According to the "Quality Standards for Veterinary Biological Products of the People's Republic of China" appendix 301, sterile growth.

[0072] Inactivation test: After inactivation, samples were taken from the virus inactivation solution under aseptic conditions, and 10 10-day-old SPF chicken embryos were inoculated via the allantoic cavity route, 0.1ml / embryo, incubated at 37°C, and observed in eggs every day, 120 There should be no death within 1 hour. If there is death, the allantoic fluid should have no hemagglutination value.

[0073] 2. Finished product inspection

[0074] Properties: The appearance is white emulsion, and the dosage form is oil-in-water-in-oil type.

[0075] Stability: Stored at 37°C for 21 days or centrifuged at 3000r / min for 15 minutes, the e...

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Abstract

The invention relates to a method for producing an avian influenza vaccine by adopting an MDCK cell line and a product of the avian influenza vaccine. The method comprises the following steps of domesticating adherent MDCK cells into the stably proliferated serum-free full-suspension culture type MDCK cell line, then performing toxin inoculation and proliferation, separating and collecting virus liquid, performing inactivation, and adding an adjuvant to prepare the vaccine. The cell line is used for replacing chick embryo tissue culture to prepare avian influenza virus and the avian influenzavaccine, so that the problem that chick embryos are polluted by exogenous virus can be solved; and by strictly controlling raw materials and culture conditions, the purity of the produced vaccine is ensured, the safety of vaccine is ensured, the method can be applied to large-scale production under safe and economic conditions, and the effective avian influenza vaccine with high purity and ideal industrial yield is obtained.

Description

technical field [0001] The invention relates to the technical field of vaccine preparation, in particular to a method for producing an avian influenza vaccine using MDCK cell lines and a product thereof. Background technique [0002] Avian influenza (Bird Flu or Avian Influenza) is an acute infectious disease caused by a subtype of influenza A virus (also known as avian influenza virus), which seriously threatens the breeding industry and human health, and brings huge losses to the national economy. So far, no ideal treatment for bird flu has been found, and vaccination is still the most effective means to prevent the occurrence and spread of the disease. [0003] There are many problems in the traditional chicken embryo technology used for the preparation of avian influenza vaccines, such as long production cycle, cumbersome operation, and heavy workload. After the virus liquid is harvested, the remaining chicken embryos need to be harmlessly treated, which is easy to be in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/145A61P31/16C12N7/00C12N7/02C12N7/06C12N5/071C12R1/93
CPCA61K39/12A61P31/16C12N7/00C12N5/0686A61K2039/552C12N2760/16134C12N2760/16151C12N2760/16163C12N2500/32C12N2500/40C12N2500/38C12N2500/22C12N2500/20C12N2500/12C12N2500/10C12N2500/36C12N2500/25C12N2500/34C12N2500/76
Inventor 施维松张晓楠詹烜子卢少华刘德城郑彩丽孔凤英
Owner ZHAOQING DAHUANONG BIOLOGIC PHARMA
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