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Optimizing process method for amplifying influenza virus H1N1 through bioreactor

A bioreactor and influenza virus technology, applied in the biological field, can solve the problem of low virus yield in a single cell, achieve good application prospects, high efficiency, and save labor and material costs.

Inactive Publication Date: 2018-05-22
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] There has been some research on the process optimization of bioreactor amplification of MDCK cells and influenza H1N1 virus. At present, the process of amplifying MDCK virus in suspension culture is basically the mainstream, and there are also microcarrier Cytodex series as bioreactor carriers to amplify influenza virus. The study of H1N1 reported that the above culture system has the advantage of being easy to scale up, but due to the high shear force of suspension culture, the virus yield of single cells is relatively low

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  • Optimizing process method for amplifying influenza virus H1N1 through bioreactor
  • Optimizing process method for amplifying influenza virus H1N1 through bioreactor
  • Optimizing process method for amplifying influenza virus H1N1 through bioreactor

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Embodiment 1

[0033] Bioreactor: China Hangzhou AMP Bioengineering Co., Ltd. 10L torrent bioreactor with a working volume of 8L, of which the cell perfusion bag volume is 4L, and the cell density is calculated as 4L. Instrument model: AP20SC;

[0034] Microcarrier: Fibra disk paper carrier 150g (Hangzhou Anpu Bioengineering Co., Ltd.);

[0035] Cells: MDCK, purchased from ATCC, USA;

[0036] Virus: Influenza H1N1 type, general vaccine strain (A / New Caledonia / 20 / 99); vaccination MOI=0.05;

[0037] Cell growth medium: DMEM medium containing 10% serum by volume (Gibco, USA);

[0038] Inoculation adsorption solution: DMEM medium (Gibco, USA) containing 0.25% hydrolyzed milk protein + 0.5% trypsin (Gibco, USA);

[0039] Cell maintenance solution: DMEM medium (Gibco, USA) containing 0.25% hydrolyzed milk protein + 0.25% trypsin (Gibco, USA);

[0040] Determination of glucose content in growth fluid:

[0041] Glucose determination kit (glucose oxidase-peroxidase method), Shanghai Rongsheng Bio...

Embodiment 2

[0055] Bioreactor: Bioflo 310 cell reactor (NBS, USA); microcarrier: Fibra disk paper carrier 150g (NBS, USA);

[0056] Virus: Influenza H1N1 type, general vaccine strain (A / New Caledonia / 20 / 99); vaccination MOI=0.05;

[0057] Cell growth medium: DMEM medium containing 10% serum by volume (Gibco, USA);

[0058] Inoculation adsorption solution: DMEM medium (Gibco, USA) containing 0.25% hydrolyzed milk protein + 0.5% trypsin (Gibco, USA);

[0059] Cell maintenance solution: DMEM medium (Gibco, USA) containing 0.25% hydrolyzed milk protein + 0.25% trypsin (Gibco, USA);

[0060] Cell culture: 150g of sterilized Fibra disk carrier has been added to the 5L reactor, soaked overnight in phosphate buffered saline solution PBS of pH 7.2, discarded and added to the cell growth medium, and inoculated with MDCK cells, the inoculum amount is (1.3±0.1 ) x 10 9 cells to be cultured. The culture parameters were set as follows: pH value 7.2-7.6, temperature 37°C, dissolved oxygen 55-80%, st...

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Abstract

The invention discloses an optimizing process method for amplifying an influenza virus H1N1 through a bioreactor. The method uses a poly-fiber (Fibra disk) carrier and a bioreactor system for amplifying a madin-darby canine kidney epithelial cell (MDCK) so as to build a whole set of process flow for replicating and amplifying the influenza virus. The method provided by the invention is built underthe condition of comprehensively adapting to an H1N1 type influenza virus replication and amplification system, a DMEM (Dulbecco Modified Eagle Medium) containing 10 percent of serum is used at a cell culture stage, the DMEM with 5 percent of serum and 0.5 percent of pancreatin are used during a period from after inoculating and adsorbing the virus to 24 hours, a serum-free medium added with 0.25percent of lactoalbumin hydrolysate and 0.25 percent of pancreatin is adopted at a virus harvesting stage, 1.5g / L of glucose is supplemented every 24 hours at the same time, and a batch type harvesting and medium changing method is adopted. According to the method, a combined medium scheme is used based on an optimizing process, so that higher virus titer can be reached, and the built optimizingprocess is the process with favorable repeatability and a high-efficient influenza virus H1N1 amplification function.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a set of optimized process methods for amplifying influenza virus H1N1 in a bioreactor used for amplifying influenza virus H1N1. Background technique [0002] MDCK cells are the packaging cells of influenza virus. The current small-scale production of influenza virus vaccines is still mainly produced in the form of chicken embryo culture, which has backward technology, cannot be further optimized, labor-intensive, and insufficient production capacity. Therefore, the large-scale bioreactor cultivation of MDCK cells and the large-scale amplification technology of influenza virus H1N1 have very important market significance. [0003] In order to improve the influenza virus H1N1 amplification process, it is necessary to have a comprehensive understanding of the physiological and growth characteristics of MDCK cells and a deep understanding of the replication mechanism of H1N1 infected ce...

Claims

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Application Information

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IPC IPC(8): C12N7/00C12N5/071
CPCC12N5/0686C12N7/00C12N2533/30C12N2760/16151
Inventor 李兰娟陈科达余东山吴晓鑫张严峻欧会林
Owner ZHEJIANG UNIV
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