CN114736846B - WAYNE293LVPRO cell culture medium additive for improving adenovirus production and preparation method thereof - Google Patents
WAYNE293LVPRO cell culture medium additive for improving adenovirus production and preparation method thereof Download PDFInfo
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- CN114736846B CN114736846B CN202210457724.3A CN202210457724A CN114736846B CN 114736846 B CN114736846 B CN 114736846B CN 202210457724 A CN202210457724 A CN 202210457724A CN 114736846 B CN114736846 B CN 114736846B
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Abstract
The invention discloses a WAYNE293LVPRO cell culture medium additive for improving adenovirus production and a preparation method thereof. It is cystine 2-6g/L, aspartic acid dipeptide 2-3g/L and lysine tripeptide 2-3g/L. The WAYNE293LVPRO cell culture medium additive can remarkably improve the adenovirus production capacity of WAYNE293LVPRO cells. The WAYNE293LVPRO cell culture medium additive is a low-cost serum-free animal-derived culture medium, is very significant in the industrialization of virus drugs, and meets the requirements of large-scale expression of gene therapeutic viruses by optimizing the process and amplifying the reactor.
Description
Technical field:
the invention belongs to the technical field of biology, and particularly relates to a WAYNE293LVPRO cell culture medium for improving adenovirus production and a preparation method thereof.
The background technology is as follows:
the sales of adenovirus drugs expressed by animal cells are rapidly increasing, and large-scale animal cell suspension culture, which is one of core technologies for commercial adenovirus production, becomes a new bottleneck for adenovirus drug development. One of the main technical limitations is that there is no commercial animal suspension medium with high efficiency per se in China, but the internationally known culture media such as SIGMA, invitrogen, hyclone, lonza and the like have a formula which is secret and is unfavorable for the optimization of the subsequent culture process. Therefore, an autonomous brand serum-free and animal-derived medium with low cost is developed, the process optimization and the reactor amplification are carried out to meet the requirement of expressing a large amount of gene therapeutic viruses, and the significance for industrialization of virus medicaments is very great.
More than 70% of the marketed viral drugs are expressed by HEK203 cell host cells. At present, WAYNE293LVPRO cells have no culture medium additive specially designed for the growth of HEK293 cells and the expression of viruses under suspension culture conditions. It is now desirable to invent a novel WAYNE293LVPRO cell culture medium additive to increase adenovirus production.
The invention comprises the following steps:
it is a first object of the present invention to provide a WAYNE293LVPRO cell culture medium additive capable of significantly increasing adenovirus production by WAYNE293LVPRO cells.
The WAYNE293LVPRO cell culture medium additive for improving adenovirus production is cystine 2-6g/L, aspartic acid dipeptide 2-3g/L and lysine tripeptide 2-3g/L.
Preferably, the WAYNE293LVPRO cell culture medium additive for improving adenovirus production is cystine 4g/L, aspartic acid dipeptide 2.5g/L and lysine tripeptide 2.5g/L.
The second object of the invention is to provide a method for preparing WAYNE293LVPRO cell culture medium additive for improving adenovirus production, which comprises the steps of adding each component into water according to the content, and sterilizing.
The WAYNE293LVPRO cell culture medium additive can remarkably improve the adenovirus production capacity of WAYNE293LVPRO cells. The WAYNE293LVPRO cell culture medium additive is a low-cost serum-free animal-derived culture medium, is very significant in the industrialization of virus drugs, and meets the requirements of large-scale expression of gene therapeutic viruses by optimizing the process and amplifying the reactor.
The specific embodiment is as follows:
the following examples are further illustrative of the invention and are not intended to be limiting thereof.
Example 1:
1. commercial AAV viral vector plasmids, packaging plasmids and helper plasmids (all available from Hexacarrier Biotechnology Co., ltd.) were used at a concentration of greater than 1ug/ul and A260/280 was between 1.7 and 1.8.
2. At 5X 10 5 Cell concentration of individual/ml WAYNE293LVPRO cells (which is disclosed in application number CN202110688920.7, title: an adaptive none)In the application of WAYNE293LVPRO cells in serum culture medium environment and application thereof, the preservation information is as follows: human embryonic kidney cells WAYNE293 were preserved in China general microbiological culture Collection center (CGMCC) at the address: beijing, chaoyang, north Chen Xi Lu 1, 3, china academy of sciences microbiological institute, postal code: 100101, deposit number: CGMCC No. 22348), shaking culture at 37 deg.C and 120rpm for 4 days until WAYNE293LVPRO cell concentration reaches 5-10×10 6 And each ml.
3. Fat-converting complex was prepared by mixing at the following ratio
4. Mu.l of lipofectex was added to 3ml of the culture medium containing WAYNE293LVPRO from step 2 and mixed well.
5. Carefully adding lipofectex mixture, shaking cross shape for 3 times, and adding 5% CO at 37deg.C 2 Under the condition, the culture is carried out in an incubator for 90 minutes, and the preculture is completed.
6. After the preculture was completed, 27ml of WAYNE293LVPRO broth (product No. A21501 of the biological technology Co., ltd., zhongshan Kang Tian) was added, and the culture was continued at 37℃with a shaking table at 120rpm for 24 hours.
After 7.24 hours, 5mL of the additive was added to the culture system, and the culture was continued for 72 hours, and then the number of virus particles was measured to estimate the virus titer. As a control, 5mL of water was added without adding 5mL of the additive. Each treatment was repeated 3 times. The additive contains an aqueous solution of 4g/L of cystine, 2.5g/L of aspartic acid dipeptide and 2.5g/L of lysine tripeptide, and is prepared by adding cystine, aspartic acid dipeptide and lysine tripeptide into water according to the content, filtering and sterilizing.
Since the viral particles are present in both the packaging cells and the culture supernatant, both the cells and the culture supernatant can be collected for best yields. A dry ice ethanol bath (ethanol is poured into a foam box filled with dry ice, or liquid nitrogen is used for replacing the dry ice ethanol bath) and a water bath at 37 ℃ are prepared. The toxigenic cells were collected along with the medium in a 15ml centrifuge tube. When collecting cells, the culture dish is tilted at an angle to scrape the cells into the medium, centrifuged at 1000rpm/min for 3 minutes, the cells and supernatant are separated, the supernatant is stored in addition, and the cells are resuspended in 1ml PBS. The cell suspension was repeatedly transferred in a dry ice ethanol bath and a 37 ℃ water bath, frozen and thawed four times. Slightly shaking after each melting. Note that: each setting and thawing takes approximately ten minutes. The number of virus particles was then measured (see methods: overview of rapid virus identification and quantitative detection methods, kumar Pankaj University of Saskatchewan, saskatoon, canada, N.J. Prinseton, inc. (Synatm Research) DOI http:// dx.doi.org/10.13070/mm.cn.3.207).
The experimental results are as follows: the adenovirus yield of the additive was 2.5X10 6 Each viral vector/ml was far above the control 0.5X10 s 6 Each viral vector/ml. It can be seen that the additive significantly increased adenovirus production by WAYNE293 LVPRO.
Example 2:
WAYNE293LVPRO cell culture medium additive, which is an aqueous solution of cystine 2g/L, aspartic acid dipeptide 3g/L and lysine tripeptide 2g/L, was prepared as in example 1.
Example 3:
WAYNE293LVPRO cell culture medium additive, which is an aqueous solution of cystine 6g/L, aspartic acid dipeptide 2g/L and lysine tripeptide 3g/L, was prepared as in example 1.
Claims (2)
1. A WAYNE293LVPRO cell culture medium additive for improving adenovirus production is characterized in that the additive is cystine 4g/L, aspartic acid dipeptide 2.5g/L and lysine tripeptide 2.5g/L.
2. A method for preparing a WAYNE293LVPRO cell culture medium additive for improving adenovirus production as claimed in claim 1, wherein each component is added into water according to the content, and the mixture is sterilized.
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| CN202210457724.3A CN114736846B (en) | 2022-04-27 | 2022-04-27 | WAYNE293LVPRO cell culture medium additive for improving adenovirus production and preparation method thereof |
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| CN114807047B (en) * | 2022-04-28 | 2022-11-15 | 中山康天晟合生物技术有限公司 | Human embryo kidney cell 293 of high expression virus and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101235365A (en) * | 2007-01-31 | 2008-08-06 | 深圳市清华源兴生物医药科技有限公司 | Highly effective method for producing adenovirus |
| CN113061584A (en) * | 2021-05-14 | 2021-07-02 | 赵峻岭 | Culture medium additive capable of promoting growth of adenovirus |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101235365A (en) * | 2007-01-31 | 2008-08-06 | 深圳市清华源兴生物医药科技有限公司 | Highly effective method for producing adenovirus |
| CN113061584A (en) * | 2021-05-14 | 2021-07-02 | 赵峻岭 | Culture medium additive capable of promoting growth of adenovirus |
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