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Culture medium used for Chinese hamster ovary cells

A technology of ovarian cells and Chinese hamsters, applied in the field of cell culture, can solve the problems of high cost, medium cost, and low protein production

Active Publication Date: 2014-07-16
SHANGHAI CELGEN BIO PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Invitrogen also produces serum-free medium, but large-scale protein expression of antibody drugs requires a large amount of medium, which is expensive. For example, the price of JRH basic medium is more than 100 yuan per liter, which accounts for a large part of the cost. ratio, in my country, protein drug manufacturers are also faced with the problem of huge cost of culture medium, and at the same time, low protein production is also a big problem

Method used

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  • Culture medium used for Chinese hamster ovary cells
  • Culture medium used for Chinese hamster ovary cells
  • Culture medium used for Chinese hamster ovary cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0106] Preparation of basic medium for CHO cells

[0107] 1. Prepare the basic medium:

[0108] 1. Ingredients

[0109] Saijin basic CHO medium (prepared according to Table 1)

[0110] NaHCO 3 1.6g / L

[0111] L-glutamine 0.4344g / L

[0112] Glucose 6.4g / L

[0113] Insulin (10mg / ml) 50ul means: 0.5mg / L

[0114] 2. Filter preparation

[0115] Ultra-pure water: The terminal resistance value of the water maker is more than 17 megohms or more. Confirm that the balance is working normally and the balance bubble has been adjusted.

[0116] Select the filter according to the volume of the culture medium, and the suitable filter material: cartridge type hydrophilic filter element or flat filter membrane.

[0117] Cartridge filter element: 0.22um, the size is suitable for the sleeve, for integrity inspection

[0118] Flat filter membrane: 0.45um, 0.22um each, fully moistened

[0119] Filter sterilization: 122°C, 30 minutes

[0120] 3. Preparation

[0121] Dissolve CHO medium, glucose and Glutamine with 90%...

Embodiment 2

[0128] Formulating feed medium for CHO cells

[0129] 1. Preparation of feeding medium:

[0130] 1. Ingredients

[0131] Saijin feed medium (prepared according to the proportion in Table 2)

[0132] Glucose 24.4g / L

[0133] 2. Filter preparation

[0134] Ultra-pure water: The terminal resistance value of the water maker is more than 17 megohms or more. Confirm that the balance is working normally and the balance bubble has been adjusted.

[0135] Select the filter according to the volume of the culture medium, and the suitable filter material: cartridge type hydrophilic filter element or flat filter membrane.

[0136] Cartridge filter element: 0.22um, the size is suitable for the sleeve, for integrity inspection

[0137] Flat filter membrane: 0.45um, 0.22um each, fully moistened

[0138] Filter sterilization: 122°C, 30 minutes

[0139] 3. Preparation

[0140] The Jiasaijin feed medium and glucose are dissolved in ultrapure water with a final volume of 90%, fully stirred for half an hour, dilut...

Embodiment 3

[0146] Cultivation of CHO cells in basic medium

[0147] Test method: batch culture (no feeding)

[0148] Test procedure: In this culture verification, no feeding is performed. After inoculation, the cell survival rate is reduced to about 50% as the culture end point.

[0149] Culture environment: 37°C, 5% carbon dioxide

[0150] Shake flask culture process: Add the filtered basal medium and JRH medium of Example 1 into two 125ml shake flasks in the ultra-clean table. Insert seeds (counted as day 0) so that the final cell density is 0.5x10 6 , The total volume is 30ml. After the cells enter the middle of the plateau phase, they begin to cool down, which makes the cells change from the growth state to express protein.

[0151] Culture results: On the seventh day of culture with Saijin medium, the cell density reached the highest: 5.3x10 6 , The survival rate is 99.1%. And the cell density cultured with JRH basal medium is 3.9x10 6 , The survival rate is 97.5%, the JRH basal medium rea...

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Abstract

The invention discloses a basal medium for Chinese hamster ovary cells. Based on the total volume of the basal medium, the basal medium contains the following basic components: 6-6.5g / L sodium chloride, 0.5- 0.5g / L potassium chloride, 0.462-1g / L sodium pyruvate, 0.288-0.4g / L calcium nitrate, 1.464-2g / L sodium bicarbonate, 5.16-15g / L D-glucose, and 0.156-0.7g / L L Disodium Hydrogen Phosphate. The invention also discloses a feeding medium for Chinese hamster ovary cells and a culture method for Chinese hamster ovary cells.

Description

Technical field [0001] The invention relates to the field of cell culture. More specifically, the present invention relates to a culture medium and corresponding culture method for Chinese Hamster Ovary (CHO) cell culture. Background technique [0002] Among eukaryotic cells, CHO (Chinese Hamster Ovary, Chinese Hamster Ovary) cells are currently the preferred system for recombinant glycosyl protein production; because it has many advantages compared with other expression systems. At present, more and more medicinal proteins have been highly expressed in CHO cells, and many CHO-expressed drugs have been put on the market, such as EPO, Enbrel, etc. [0003] Serum-free medium is currently widely used in CHO cell culture, because medium containing animal serum has many disadvantages. Due to the limited function of serum-free medium for promoting cell growth and expression, feeding technology has now become one of the focuses of competition among major biopharmaceutical companies. [0...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071
Inventor 陈智胜
Owner SHANGHAI CELGEN BIO PHARMA CO LTD
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