Optimized technological method for amplifying recombinant adenovirus by using bioreactor
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A bioreactor, recombinant adenovirus technology, applied in biochemical equipment and methods, microorganisms, viruses/phages, etc., to achieve the effects of saving manpower and material costs, improving productivity, high repeatability and stability
Active Publication Date: 2013-06-19
ZHEJIANG PUKANG BIOTECH
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Hydrolyzed milk protein is a safe and effective medium additive, which has been widely used. There is no report on the amplified adenovirus. How to determine the concentration ratio of it is a direction worth exploring
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Embodiment 1
[0028] 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;
[0029] Microcarrier: Fibra disk paper carrier 150g (Hangzhou Anpu Bioengineering Co., Ltd.);
[0030] Cells: HEK293, purchased from ATCC, USA;
[0031] Virus: Recombinant type 5 adenovirus vector carrying human papillomavirus oncogene E6 and E7 (AdE6E7) fusion protein gene, self-constructed; inoculation MOI between 20-50;
[0032] Cell growth medium: DMEM medium containing 10% serum by volume (Gibco, USA);
[0033] Cell maintenance solution: DMEM medium (Gibco, USA) containing 0.25% hydrolyzed milk protein (Gibco, USA);
[0034] Determination of glucose content in growth fluid:
[0036] Mix 1ml of the reagents marked R1 and R2 i...
Embodiment 2
[0054] Bioreactor: Bioflo 310 cell reactor (NBS, USA); microcarrier: Fibra disk paper carrier 150g (NBS, USA);
[0055] Virus: recombinant adenovirus type 5 vector; inoculation MOI between 20-50;
[0056] Cell growth medium: DMEM medium containing 10% serum by volume (Gibco);
[0057] Cell maintenance solution: DMEM medium containing 0.25% hydrolyzed milk protein (Gibco);
[0058] Cell culture: 150g of sterilized Fibra disk carrier has been added to the 5L reactor, soaked overnight in phosphate buffer solution PBS with pH 7.2, discarded and added to cell growth medium, and inoculated with HEK293 cells, the inoculum size is (1.5±0.2 ) x 10 9 cells to be cultured. The culture parameters were set as follows: pH value 7.1-7.5, temperature 37°C, dissolved oxygen 50-80%, stirring speed 60rpm. Samples were taken regularly every day to measure glucose consumption, so as to estimate cell growth. Under stable conditions, glucose consumption was linearly related to cell density. The...
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Abstract
The invention relates to the biotechnology field and in particular relates to a comprehensively optimized technological method for amplifying recombinant adenovirus. The method is used for amplifying human embryonic kidney cells (HEK293) by using poly-fiber paper carriers through a bioreactor; in this way, the whole set of process flow of adenovirus replication and amplification is established. The optimized technological method provided by the invention comprises the following steps of: under the condition of comprehensively adapting to type 5 adenovirus replication and amplification system, utilizing a DMEM culture medium containing 10% of blood serum at the cell culture stage, utilizing a DMEM culture medium containing 20% of blood serum in 20 hours after virus inoculation absorption; employing a blood serum-free culture medium added with 0.25% of lactoalbumin hydrolysate at the virus collecting stage, and simultaneously, supplementing 2g/L glucose every 20 hours. The way of batch collection and batch liquid exchange is adopted; and the method enables the optimized process to achieve high virus titer on the basis of reducing the later-stage purification difficulty and meeting the requirements of biological products. Therefore, the optimized process established by the invention an efficient recombinant adenovirus amplification process having excellent repeatability, and is suitable for any bioreactor with the poly-fiber paper as the carriers to amplify the recombinant adenovirus.
Description
technical field [0001] The invention relates to the field of biotechnology, in particular to a set of optimized process methods for amplifying recombinant adenovirus in a bioreactor used for amplifying recombinant adenovirus. Background technique [0002] Human embryonic kidney (HEK293) cells are packaging cells for adenovirus vectors. Adenoviruses are popular vectors for gene therapy research after retroviruses. Adenovirus vectors are directly introduced into the human body to express target genes for the treatment of malignant tumors and cardiovascular diseases. Diseases or some genetic diseases have made gratifying progress; the use of adenovirus vectors to express secreted proteins in packaging cells HEK293, such as protein tyrosine kinase 1C, has also become a way to produce recombinant proteins. In 2004, the world's first gene therapy product "Jinyousheng" was launched in China, which enabled the real application of adenovirus vectors in clinical practice. Therefor...
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