Chemically-defined medium, application thereof and production technology for large-scale culture of mammalian cells

A large-scale culture and mammalian technology, which is applied in the production process of medium with defined chemical composition and large-scale culture of mammalian cells, can solve the problems of inapplicability to large-scale production, high price of medium with defined chemical composition, high production cost, etc. problem, achieve the effect of shortening development time, reducing production cost and increasing output

Active Publication Date: 2014-05-07
上海多宁生物科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These chemically defined media have their own advantages, such as better support for cell growth, but at the same time have limitations: (1) All media are specific cultures for a specific cell line or cell line For example, CD CHO medium is mainly used for CHO K1 cells, and CD OptiCHO is mainly used for DG44 cells; (2) The medium with chemical composition is expensive and the production cost is high, so it is not suitable for large-scale production
[0010] However, in the current domestic market, there is no chemically defined medium that can support high-density cell culture.

Method used

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  • Chemically-defined medium, application thereof and production technology for large-scale culture of mammalian cells
  • Chemically-defined medium, application thereof and production technology for large-scale culture of mammalian cells
  • Chemically-defined medium, application thereof and production technology for large-scale culture of mammalian cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1: the development of culture medium formula

[0049] A typical mammalian cell culture medium contains 50-70 components. Fully optimizing these components is often time-consuming and complex. Optimizing each component one by one takes a lot of time and does not achieve the effect of overall optimization. Our plan is to first divide the main components of the medium into amino acids, vitamins, trace elements, inorganic salts, antioxidants, etc., optimize the basal medium through Design of Experiments (DOE), balance all nutritional components and base on cell metabolism requirements Optimize component concentrations. A typical CHO cell culture medium formulation is shown in Table 2.

[0050] Table 2 Typical CHO cell culture medium and formula

[0051]

[0052]

[0053]

[0054] *Added when the culture medium is prepared.

Embodiment 2

[0055] Embodiment 2: the preparation of production and fed-batch feeding medium

[0056] The preparation methods of 1 liter of production medium and 1 liter of fed-batch feed medium are shown in Table 3 and Table 4, respectively.

[0057] The preparation of table 31 liters of production medium

[0058]

[0059] Table 41 Preparation of elevated concentration feeding medium

[0060]

[0061]

Embodiment 3

[0062] Example 3: Cultivate different types of CHO cells (CHO K1, CHO S and CHO DG44) using the medium of the present invention (production medium in Example 2)

[0063] The cryopreservation tubes of CHO K1, CHO S and CHO DG44 cells were taken out from the liquid nitrogen tank respectively, and immediately revived in a 37°C water bath. Aseptically transfer to 10 mL of fresh medium to resuspend the cells. After counting, dilute the culture volume to 30mL and transfer to a 125ml shake flask for culture, the initial cell concentration is (0.5-1)×10 6 cells / mL, culture conditions are 37℃, humidity ≥85% and 5% CO 2 , samples were taken every day, and the cell density and viability were recorded.

[0064] from figure 1 It can be seen that all three kinds of cells can grow in the chemically defined medium of the present invention, and the peak cell density can reach (5-6)×10 6 cells / mL, the viability was maintained above 90% after 6-7 days of culture.

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Abstract

The invention discloses a chemically-defined medium. A disclosed DMEM / F12 formula is taken as a basis, the concentration of various components is adjusted, certain components are added or eliminated on the basis, and the chemically-defined medium is applicable to the large-scale production of various mammalian cells. The invention further discloses application of the medium in large-scale culture of mammalian cells (CHO cells, BHK cells or hybridoma cells) and a production technology for large-scale culture of mammalian cells. The chemically-defined medium provided by the invention does not contain serum or proteins, eliminates animal ingredients and vegetable protein hydrolysate used in the conventional medium, and facilitates the product purification and production batch stability, and the chemical constituents of all the components are defined, so that the product quality is guaranteed and improved; through the adoption of the medium and the production technology, provided by the invention, the cell density is improved, and the yield of target products is increased, and the overall production cost is low.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a culture medium with defined chemical composition, its application and a production process for large-scale culture of mammalian cells. Background technique [0002] With the advent of recombinant DNA technology, culture media have been widely used in a variety of cell cultures, including animals, plants and bacteria. Target products in the biopharmaceutical industry, such as antibodies, can be produced in large quantities by culturing cells in liquid media. These cells, either natural or engineered, rapidly multiply in liquid media using bioreactors and produce the desired product. These products can be collected directly from the culture medium or extracted from harvested cells. [0003] The medium provides the nutrients needed for the cell culture process. In the early cell culture process, the medium formula was based on the chemical composition and physicochemical properties ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12P21/08
Inventor 李锋叶培
Owner 上海多宁生物科技股份有限公司
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