Method of bioreactor micro-carrier for cultivating human diploid cell to produce viral vaccine

A technology of human diploid cells and bioreactors, applied in biochemical equipment and methods, animal cells, vertebrate cells, etc., can solve the problem of limited number of passages, high requirements for growth environment, and large-scale cultivation of difficult bioreactors, etc. problems, to achieve high output, good consistency, and large-scale industrial production

Inactive Publication Date: 2012-07-11
BEIJING SKYWING TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The use of bioreactor microcarriers to culture animal cells for the production of biological products is an inevitable trend in the biopharmaceutical industry. However, due to the slow growth of human diploid cells, the limited number of passages, and high requirements for the growth environment, it is generally considered difficult to carry out large-scale cell culture. , especially difficult to achieve large-scale cultivation using bioreactors

Method used

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  • Method of bioreactor micro-carrier for cultivating human diploid cell to produce viral vaccine
  • Method of bioreactor micro-carrier for cultivating human diploid cell to produce viral vaccine
  • Method of bioreactor micro-carrier for cultivating human diploid cell to produce viral vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0115] Hydrate the microcarriers and sterilize the bioreactor holding the hydration microcarriers in advance as follows:

[0116] Mix the microcarriers with 50 mL / g of Ca-free 2+ and Mg 2+ Mix with PBS solution, start hydration at room temperature, and stir at 50 rpm for 2 minutes every 15 minutes. After hydration overnight, stop stirring, settle naturally, discard the supernatant, and complete the pretreatment;

[0117] Add 30 mL / g of fresh Ca-free 2+ and Mg 2+ Then stir for 2 minutes at a speed of 50 rpm every 15 minutes, stop stirring after 30 minutes, settle naturally, discard the supernatant, and complete the washing; repeat the washing once, and discard the supernatant PBS used for washing;

[0118] Add 30 mL / g of fresh Ca-free 2+ and Mg 2+ The PBS liquid, obtains the microcarrier of hydration;

[0119] Then place the hydrated microcarrier in the tank of the reactor; connect the various pipelines of the reactor, and sterilize at 121° C. for 30 minutes;

[0120] As...

Embodiment 2-4

[0140] According to the conditions described in Table 1, according to the same operation as Example 1, Examples 2-4 were carried out to obtain supernatant virus liquid.

[0141] Table 1

[0142]

Embodiment 5

[0144] This example is used to illustrate a more specific method for producing virus vaccines by culturing human diploid cells with microcarriers in a bioreactor of the present invention.

[0145] 1) Recovery of MRC-5 cells (purchased from ATCC):

[0146] Immediately thaw the frozen MRC-5 cells taken out of liquid nitrogen into a 37°C water bath;

[0147] Add 1ml of frozen cell solution to 5ml of fresh cell culture medium and mix gently;

[0148] Centrifuge at 800 rpm for 5 minutes, discard the supernatant;

[0149] Add 5ml of fresh cell culture medium to the cell pellet and mix gently;

[0150] Centrifuge again at 800rpm for 5 minutes, discard the supernatant;

[0151] Add 5ml of fresh cell culture medium to the cell pellet, mix gently, transfer to a culture flask, add cell growth medium to 20ml, and store at 37°C, 5% CO 2 Cultivate in the incubator until the cells grow flat on the bottom of the flask;

[0152] 2) Digest MRC-5 cells:

[0153] Pour off the culture medium...

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Abstract

The utility model provides a method of bioreactor micro-carrier for cultivating human diploid cell to produce viral vaccine. The method comprises the following steps: reviving human diploid cell; amplifying the culture of the human diploid cell and preparing the cell suspension; inoculating the obtained cell suspension to the bioreactor, fresh complete cell growth culture solution for sterilizing and micro-carrier are pre-added into the micro-carrier, the final density of the inoculated cell is 5*104-2*106 cell / ml, the monolayer cell of the micro-carrier is cultivated under the conditions that the temperature is 35 to 39 degrees, pH is 6.5 to 8.0, and the dissolved oxygen is 15 to 100%, and the cell density amounts to more than 1*106 cell / ml; and inoculating, breeding and harvesting virus. According to the method, utilizing the bioreactor for cultivating the human diploid cell to produce the virus vaccine in large scale becomes possible, the produced virus vaccine has high output and consistency, and the large-scale industrial production for the virus vaccine is effectively realized.

Description

technical field [0001] The invention relates to a method for producing a virus vaccine, in particular, the invention relates to a method for producing a virus vaccine by culturing human diploid cells in a bioreactor microcarrier. Background technique [0002] Human diploid cells refer to cells derived from normal human somatic cells, which can be subcultured in vitro and have a wide range of sensitivity to a variety of viruses. Human diploid cells are not tumor cells, so they have a certain stable passage life (usually 40-60 passages), beyond which the aging, degeneration and death of cells may occur. Within the subculture lifespan, the karyotype of any subculture level of human diploid cells maintains the normal number of diploid chromosomes, and the number of diploid cells should account for more than 75% of the total number of metaphase cells examined. [0003] Although human diploid cell lines have a limited lifespan in stable passage, they can freeze excess cells at an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/08C12N5/071C12N11/00
Inventor 张韧陈文庆王建超
Owner BEIJING SKYWING TECH CO LTD
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