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Supplementing culture medium for CHO cell culture and preparing method and application thereof

A technology for feeding medium and cell culture, which is applied to the field of feeding medium for Chinese hamster ovary cell culture and its preparation, can solve problems such as unmet demand, achieves simple preparation method, broad application prospect, and maintains cell viability. rate effect

Active Publication Date: 2019-10-18
TONGHUA DONGBAO PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Although some feed media as described above are available in the prior art, biopharmaceutical companies have an unmet need for low cost, high protein yield feed media

Method used

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  • Supplementing culture medium for CHO cell culture and preparing method and application thereof
  • Supplementing culture medium for CHO cell culture and preparing method and application thereof
  • Supplementing culture medium for CHO cell culture and preparing method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0079] Example 1 A kind of feeding medium for CHO cell culture

[0080] The feed medium for CHO cell culture described in this example, the raw material composition is shown in Table 1, prepared by the following method:

[0081] 1) According to the preparation total mass is 1000g, weigh various amino acids, vitamins and sodium hydroxide according to the ratio shown in Table 1;

[0082] 2) adding sodium hydroxide to water for injection to dissolve, set the volume to 5mol / L, and cool to room temperature to obtain sodium hydroxide solution;

[0083] 3) Take another 550-600g of water for injection, add 90% of the total amount of sodium hydroxide solution prepared in step 2), and stir evenly;

[0084] 4) Add the amino acid and vitamin into the solution prepared in step 3), and keep stirring for 30-60min;

[0085] 5) Slowly add the remaining sodium hydroxide solution prepared in step 2), and stir until the powder is completely dissolved;

[0086] 6) Dilute to 1000g with the rem...

Embodiment 2～6

[0089] Embodiment 2~6 A kind of feeding medium for CHO cell culture

[0090] For the feed medium for CHO cell culture described in Examples 2 to 6 above, the raw material composition is shown in Table 1, and 1000 g of the feed medium were prepared according to the same steps as in Example 1. See Table 3 for the test results of pH value, turbidity and osmotic pressure of the feeding medium in each embodiment. After testing, all the above indicators meet the requirements.

Embodiment 7～25

[0099] Embodiment 7-25 CHO cell culture

[0100] Basal medium: commercially available, see Table 4 for details;

[0101] Feed medium: the feed medium prepared in Examples 1-6, commercially available feed medium (see Table 4 for details);

[0102] Cell line: 1) CHO-K1 cells encoding antibody-1, wherein the antibody-1 refers to Example 1 of the Chinese patent ZL200580026569.4 specification; 2) CHO-K1 cells encoding antibody-2, wherein the antibody -2 Refer to Example 1 of Chinese Patent ZL 200980143007.6; 3) CHO-K1 cells containing a fusion protein, wherein the fusion protein refers to Example 1 of Chinese Patent ZL 200480018573.1.

[0103] Cell culture was carried out as follows:

[0104] I) According to 0.55±0.10(×10 6 cells / ml), CHO-K1 cells were inoculated into a 250ml shake flask, and the basal medium was shown in Table 4; the initial culture volume was 80ml, the culture temperature was 36.5±0.5°C, the shaker speed was 120±10rpm, and the carbon dioxide concentration 8...

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Abstract

The invention relates to a supplementing culture medium for CHO cell culture and a CHO cell culture method. The supplementing culture medium comprises amino acids, vitamins, sodium hydroxide and waterfor injection. According to the total mass of the supplementing culture medium, the supplementing culture medium comprises, by mass, 4.8-19.75% of amino acids, 0.00925-0.13% of vitamins, 1.25-5.5% ofsodium hydroxide and the balance water for injection, wherein the amino acids include L-aspartic acid, L-tyrosine, L-threonine, L-tryptophan and L-glutamic acid, and the vitamins include riboflavin,folic acid, vitamin B12, biotin, menadione sodium bisulfite, thiamine hydrochloride and L-ascorbic acid. By means of the supplementing culture medium and the cell culture method, CHO cell growth can be optimized, and the capacity of cells of expressing target protein can be improved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a feeding medium for Chinese hamster ovary cell (CHO cell) culture, a preparation method and application thereof. Background technique [0002] CHO cells (Chinese Hamster Ovary Cells, Chinese Hamster Ovary Cells) are a widely used mammalian cell expression system. Most of the therapeutic monoclonal antibodies that have been marketed and entered the clinical and preclinical research stages are produced using this expression system. The medium is the material basis for the growth and product expression of CHO cells. According to the timing and purpose of addition, the medium can be divided into basal medium and feed medium, in which the basal medium is mainly used in the early proliferation stage of cells, and the feed medium is mainly used in the early proliferation and expression stage. Because the culture medium containing animal serum (such as fetal bovine serum, etc.)...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12P21/08C12P21/00
CPCC12N5/0682C12P21/00C12N2500/32C12N2500/12C12N2500/38C12N2510/00
Inventor 王德朋王明焱李美娇钱瑞鹏冷春生常晓慧
Owner TONGHUA DONGBAO PHARMA
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