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Highly effective method for producing adenovirus

A technology of adenovirus and recombinant adenovirus, applied in biochemical equipment and methods, virus/bacteriophage, botanical equipment and methods, etc., can solve the problems of easy pollution, no description of cultivation method, complicated purification, etc., and achieve low cost, Suitable for industrial production, high output effect

Inactive Publication Date: 2008-08-06
TSINGHUA YUANXING BIO PHARM SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] None of the above methods have explained the specific cultivation method
At the same time, the production of adenovirus by collecting cells and breaking the suspension method has the following disadvantages: when the cells are broken, it is easy to produce aerosol, and it is easy to pollute the virus that can spread in the air; Purification is more complicated

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1. Production of CHB3 recombinant adenovirus

[0028] Materials: a) Cells: HEK293 cells (human embryonic kidney cells)

[0029] b) Culture medium: cell culture medium: DMEM (GIBCO company) + 10% CCS (HYCLONE company), virus culture medium: DMEM + 2% CCS

[0030] c) Virus: CHB3 is a genetically engineered, replication-incompetent recombinant human adenovirus type 5.

[0031] d) Reactor and carrier: The reactor used was a 5L bioreactor (bioflo3000universal reactor, NBS, USA), and the working volume was 3.5L. The carrier used for adherent culture was DISK carrier (NBS Corporation, USA) 150g.

[0032] method:

[0033] a) Seeding and culturing cells: The cell culture medium is first added to the bioreactor. When the operating conditions were stable at 37°C, PH7.2, DO 62.1% (dissolved oxygen), 60rpm (revolution / min), HEK293 cells were inoculated into the bioreactor at a density of 5×10 5 cells / ml,. The cell culture conditions are temperature 37°C, rotation speed...

Embodiment 2

[0036] Example 2. Production of CHB3 recombinant adenovirus

[0037] The production steps were the same as those in Example 1, except that the carrier used for adherent culture was 100 g of DISK carrier, the MOI was 5, and the cell culture medium was DMEM+2% serum. The virus culture medium is DMEM+0.01% serum. The cell seeding density was 2.5 × 10 5 cells / ml, and the glucose concentration was maintained at about 0.1 g / l after virus inoculation.

Embodiment 3

[0038] Example 3. Production of CHB3 recombinant adenovirus

[0039] The production steps are the same as those in Example 1, except that the carrier used for adherent culture is DISK carrier 150g, the MOI is 50, and the cell culture medium is DMEM+5% serum. The virus culture medium is DMEM+1% serum. Cell seeding density is 5 × 10 5 cells / ml, and the glucose concentration was maintained at about 1.6 g / l after virus inoculation.

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PUM

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Abstract

The invention relates to a method for producing adenovirus hominis, the method comprises the following steps: inoculating host cell, leading cells to grow in culture medium, replacing culture solution in a bioreactor, utilizing adenovirus hominis to infect the host cell, breeding virus, gathering virus suspension liquid and concentrating through monitoring virus concentration in the bioreactor, and the method also comprises utilizing chromatography to separate adenovirus hominis. The producing method can produce adenovirus hominis which is in accordance with the requirement of clinical medication in large scale. The method has the advantages of high yield, large scale and low production cost, which is suitable for industrial production.

Description

technical field [0001] The present invention relates to the field of biopharmaceutical technology, in particular to a method for producing recombinant adenovirus. Background technique [0002] With the launch of the world's first gene therapy product Jinshengsheng in 2004, gene therapy has been increasingly recognized by people. Adenovirus is a commonly used gene virus vector in gene therapy. Its advantages are high transfection efficiency, good operability, it can carry larger target gene fragments, and it can prepare high-titer virus particles. It is easy for industrial production and can infect both Dividing cells can also infect non-dividing cells, with the advantages of safety and low pathogenicity. Therefore, it is very important to produce high-quality and large quantities of adenoviruses that meet the clinical drug requirements. [0003] The traditional method of mass production of adenovirus is tissue culture flasks or "cell factories". Virus-infected cells are h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C12N15/861A61K48/00
Inventor 董艳荣郭卫华石志斌黄伟东彭忞曾小平敖然秦华周向军
Owner TSINGHUA YUANXING BIO PHARM SCI & TECH
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