Method for producing avian influenza vaccine by using WAVE bioreactor

A technology of bioreactor and bird flu, which is applied in the direction of pharmaceutical formulations, medical preparations containing active ingredients, antibody medical ingredients, etc., can solve the problems of exogenous virus infection, high labor intensity, bacterial contamination, etc., and achieve the improvement of cultivation efficiency , the effect of improving production efficiency

Active Publication Date: 2013-11-20
黑龙江省百洲生物工程有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] At present, bird flu vaccines are mainly produced from chicken embryos. This traditional production method is labor-intensive, the source of chicken embryos is unstable, and a cumbersome purification process is required to remove chicken embryo proteins to reduce allergic reactions. At the same time, t

Method used

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  • Method for producing avian influenza vaccine by using WAVE bioreactor
  • Method for producing avian influenza vaccine by using WAVE bioreactor
  • Method for producing avian influenza vaccine by using WAVE bioreactor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Embodiment 1 utilizes WAVE wave bioreactor to produce bird flu vaccine

[0018] 1. Cell Culture

[0019] 1.1 Microcarrier treatment: Weigh an appropriate amount of microcarrier (Cytodex-1) according to the final volume of culture, and use Ca-free 2+ , Mg 2+ -Soak overnight in PBS at room temperature, discard the PBS, and use Ca-free 2+ , Mg 2+ -Wash once with PBS, discard, and finally add Ca-free 2+ , Mg 2+ - PBS autoclaved (115 °C, 10 psi, 15 min).

[0020] 1.2 Cell recovery culture: Use a square bottle to culture MDCK cells recovered from a liquid nitrogen tank. The culture conditions include: pH value 7.2, temperature 37°C; culture for 48-72 hours, and when a good cell monolayer is formed, it is used for further passage or inoculation in Microcarrier suspension culture was carried out in a wave bioreactor; the medium used in this process was DMEM, and the serum was fetal bovine serum, and the usage amount was 10%.

[0021] 1.3 Microcarrier culture: prepare MDC...

experiment example 1

[0026] The optimization experiment of experimental example 1 microcarrier content

[0027] Cell inoculation density is closely related to the influence of microcarrier content on cell growth. The important sign is to ensure the appropriate ratio of cell density to carrier during inoculation; in order to produce high-titer virus, a higher cell density is required. At the same time, In order to culture cells faster and more efficiently, it is required that the cells have a higher cell expansion factor.

[0028] Under the premise of inoculating the same number of MDCK cells per gram of microcarrier, when the Cytodex1 content was 1g / L, 2g / L, 3g / L, 4g / L, 5g / L, 6g / L, 7g / L, 8g / L The growth of MDCK cells is used to determine the appropriate carrier dosage.

[0029]The experimental results are shown in Table 1 and Table 2. It can be seen from Table 1 and Table 2 that with the increase of the microcarrier content, the MDCK cell density will increase, but the MDCK cell expansion ratio ...

experiment example 2

[0035] Experimental example 2 Screening experiment of MDCK cell seeding density

[0036] When the amount of microcarriers is 2g / L, 5×10 4 , 1×10 5 , 2×10 5 , 3×10 5 , 4×10 5 , 5×10 5 , 6×10 5 MDCK cells were inoculated at an inoculation density of 1 / mL, and samples were taken and analyzed every day. The growth of MDCK cells is shown in Table 3.

[0037] Table 3 Growth of MDCK cells at different seeding densities (unit / mL)

[0038]

[0039] It can be seen from Table 3 that when the content of microcarriers is equal, different seeding densities have a greater impact on the growth of MDCK cells. Take 5×10 4 , 1×10 5 Individual / mL inoculation, the inoculation density was low and it was still growing slowly 5 days after inoculation, and there was no obvious sign of entering the stable growth period; with 2×10 5 , 3×10 5 , 4×10 5 , 5×10 5 , 6×10 5 Cells / mL was inoculated with a high inoculation density, and MDCK cells entered a stable growth phase on the second day ...

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Abstract

The invention discloses a method for producing an avian influenza vaccine by using a WAVE bioreactor, which comprises the following steps: (1) cell culture: inoculating cells onto a micro supporter of a cell culture bag, adding a cell culture medium, and shaking the cell culture bag to perform cell culture; (2) virus solution multiplication: settling the micro supporter, washing the cultured cells, inoculating with an avian influenza virus seed, adding a virus multiplication culture medium, and further performing cell culture, thus obtaining a virus solution; and (3) inactivating the virus solution, and emulsifying to obtain the avian influenza vaccine. The method disclosed by the invention optimizes the technological parameters in the process of culturing MDCK cells by using a WAVE bioreactor, effectively improves the MDCK cell culture efficiency, and finally increases the avian influenza vaccine production efficiency remarkably. The immunoprotection efficacy experiment proves that the avian influenza vaccine prepared by the method disclosed by the invention has exact immunoprotection efficacy for poultry.

Description

technical field [0001] The invention relates to a production method of an avian influenza vaccine, in particular to a method for producing an avian influenza vaccine by using a WAVE wave bioreactor, and belongs to the production field of avian influenza vaccine. Background technique [0002] At present, bird flu vaccines are mainly produced from chicken embryos. This traditional production method is labor-intensive, the source of chicken embryos is unstable, and a cumbersome purification process is required to remove chicken embryo proteins to reduce allergic reactions. At the same time, there are unavoidable bacteria There are many problems such as pollution and foreign virus infection, and the use of bioreactors instead of chicken embryos to produce vaccine strains overcomes the above-mentioned shortcomings and becomes one of the most promising avian influenza vaccine production technologies. [0003] WAVE wave bioreactor is a new type of reactor that adopts disposable pro...

Claims

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Application Information

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IPC IPC(8): A61K39/145A61P31/16
Inventor 乔云龙张娇娇李山峰李立莉李静
Owner 黑龙江省百洲生物工程有限公司
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