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Suspension culture production method for porcine circovirus 2-type cells

A porcine circovirus, cell suspension technology, applied in microorganism-based methods, biochemical equipment and methods, viruses/phages, etc., can solve the problem of high labor, site and raw material costs, difficulty in meeting vaccine requirements, and difficulty in expanding production, etc. problem, to achieve the effect of simple and stable production process, expansion of production scale, and short production cycle

Active Publication Date: 2012-09-26
成都史纪生物制药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] At present, the production of porcine circovirus type 2 inactivated vaccine in China is mainly realized by the method of cell spinner bottle culture. The semi-finished product antigen titer produced by this traditional process is low, only 10 4.5-5.5 TCID 50 / ml, it is difficult to meet the requirements of vaccine matching (antigen content per ml should be ≥10 5.5 TICD 50 ), different times of concentration are required
And this method is labor-intensive, requiring 100-200 spinner bottles to produce a batch; time-consuming, 6-7 days; low efficiency, only one harvest per cultivation; high production cost, large labor, site and raw material costs; Polluted by the environment, the toxic waste water produced by bottle washing needs special treatment; the difference between different production batches or between different bottles of the same production batch is large, which affects the quality of the whole batch of antigens; it is difficult to expand production, requiring a large site and GMP workshop; Bottle-by-bottle inoculation and harvesting are prone to hidden dangers of vaccine quality caused by bacteria or other virus contamination, involving biosafety and public health issues

Method used

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  • Suspension culture production method for porcine circovirus 2-type cells
  • Suspension culture production method for porcine circovirus 2-type cells

Examples

Experimental program
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Effect test

Embodiment 1

[0035] A kind of porcine circovirus type 2 cell suspension culture production method, comprises the steps:

[0036] (1), Subculture and cultivation of healthy cells

[0037] The pK-15 clone cells were digested and passaged with trypsin digestion solution, cultured in cell growth medium at a temperature of 37.0°C (±0.2)°C; when a good cell monolayer was formed, it was used for further passage or inoculated in a bioreactor microcarrier culture;

[0038] (2), preparation and passage of toxic cells

[0039] Digest and passage the healthy pK-15 clone cells with good cell condition and grow into a single layer, then inoculate PCV2 seed virus according to 5% of the cell culture volume, culture for 48 hours, and after the cells grow to more than 50%, change the maintenance solution containing MEM and continue to maintain for 48 hours. After the cells are full, the F1 generation of toxic cells can be obtained; gently wash the F1 generation of toxic cells with preheated sterile PBS bu...

Embodiment 2

[0061] A kind of porcine circovirus type 2 cell suspension culture production method, comprises the steps:

[0062] (1), Subculture and cultivation of healthy cells

[0063] The pK-15 clone cells were digested and passaged with trypsin digestion solution, cultured in cell growth medium at a temperature of 37.0°C (±0.2)°C; when a good cell monolayer was formed, it was used for further passage or inoculated in a bioreactor microcarrier culture;

[0064] (2), preparation and passage of toxic cells

[0065] Digest and passage the healthy pK-15 clone cells with good cell condition and grow into a monolayer, then inoculate PCV2 seed virus according to 1% of the cell culture volume, culture for 24 hours, and after the cells grow to more than 50%, change the maintenance solution containing MEM and continue to maintain for 48 hours. After the cells are full, the F1 generation of toxic cells can be obtained; gently wash the F1 generation of toxic cells with preheated sterile PBS buffe...

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Abstract

The invention discloses a suspension culture production method for porcine circovirus 2-type cells; the method provided by the invention comprises the production technology of preparing porcine circovirus 2-type inactivated vaccines by suspension culture for poisoned cells through porcine circovirus 2-type virus biological reactor micro carriers. The method provided by the invention can greatly reduce production cost and increase output-input ratio by 5-10 times; and the method shortens production period by 2-3 days and occupies small places, wherein occupied area is only 1 / 5 of the occupied area in the traditional roller bottle process under the same yield; scale of production is enlarged fast and easily; pollution is little and processes are performed easily under the totally-enclosed tank production condition; degree of automation is high; employee is less; quality stability is realized easily; and the method can significantly reduce production cost and increase yield and quality of vaccines.

Description

technical field [0001] The invention relates to a cell culture production method, in particular to a method for producing porcine circovirus type 2 vaccine antigen using bioreactor microcarrier cell culture technology. Background technique [0002] At present, the production of porcine circovirus type 2 inactivated vaccine in China is mainly realized by the method of cell spinner bottle culture. The semi-finished product antigen titer produced by this traditional process is low, only 10 4.5-5.5 TCID 50 / ml, it is difficult to meet the requirements of vaccine matching (antigen content per ml should be ≥10 5.5 TICD 50 ), different times of concentration are required. And this method is labor-intensive, requiring 100-200 spinner bottles to produce a batch; time-consuming, 6-7 days; low efficiency, only one harvest per cultivation; high production cost, large labor, site and raw material costs; Polluted by the environment, the toxic waste water produced by bottle washing nee...

Claims

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Application Information

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IPC IPC(8): C12N7/00C12R1/93
Inventor 徐宏军丁光星任丽赵英杰胡来根刘玉才
Owner 成都史纪生物制药有限公司
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