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A kind of cho cell combined carbohydrate feed medium

A feeding medium and cell culture technology, applied in the fields of biology and CHO cell culture medium, can solve the problems of no related reports, unfavorable cell state of by-products, no reflection of temperature changes and sugar concentration, etc., to achieve enhanced production and activity, good protein stability, and improved expression efficiency

Active Publication Date: 2021-05-18
SHANGHAI OPM BIOSCI CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the above-mentioned prior art does not reflect the relationship between temperature change and sugar concentration. In the prior art, the change of sugar concentration is mainly due to a simple improvement compensation in nutritional supplements, which tends to produce more by-products that are not conducive to the state of cells.
Based on the correlation between temperature change nodes and different sugar combinations, there are no related reports

Method used

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  • A kind of cho cell combined carbohydrate feed medium
  • A kind of cho cell combined carbohydrate feed medium
  • A kind of cho cell combined carbohydrate feed medium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030]Example 1 Media Configuration Combination Gradient Medium - High Concentration Medium - Commercial Properties

[0031]10 contrecoted medium were configured in a table proportion, and the medium volume was replenished with deionized water, and the sterilization was filtered.

[0032]Table 4-1 Comparative Example 1 (unit mg / L)

[0033] Replenishment 1Replenish 2Replenishment 3Replenishment 4Replenishment 5 30℃34℃37℃34℃32℃ glucose10003000800030002000 Galactose2005003000800600 maltose5001000200016001500 Manno5005005008001000 Glucan15001800200020002000 starch800700600500400 Linolenic acid0.080.080.080.080.08 Linoleic acid0.10.10.10.10.1 cholesterol1.81.81.81.81.8 Peanut0.0050.0050.0050.0050.005 Progesterone0.10.10.10.10.1 Palmitic acid0.180.180.180.180.18 insulin0.030.030.030.030.03 Glycine5050505050 L-isoleucine120120120120120 L-leucine130130130130130 L-histidine7878787878 L-lysine256256256256256 L-methionine108108108108108 L-phenylalanine6868686868 L-threonine123123123123123 L-tryp...

Embodiment 2

[0050]Take the project CHO cell strain in a 37 ° C water bath to be thawed, and then mixed into the centrifuge tube containing the commercial resuscitation medium, 1000 rpm centrifuge 5 min carefully discarded the supernatant, take 30-50ml of the biochemical basis medium rehabilitation after CO2 culture In the case of shaker in the box for 3-5 days, sampling detection of living cell density and cell activity, to the cell density to 5 × 105When the cell activity is greater than 85%, it can be accessed to enlarge the culture vessel. The gene product of anti-PD-1 specific antibody was cloned into a mammalian expression vector PTT5, and transient expression in CHO cells (Raymond C. Methods. 55 (1): 44-51 (2011)).

[0051]The composition of the ratio 1-10 medium complement 1-5 was used to cultivate CHO cells (divided 6) (except for the Comparative Example 6), after the end of the end of the end, the culture liquid was collected from the end of the culture. Torry, use protein A box (Porosa20...

Embodiment 3

[0067]Example 3: Other various antibody expression

[0068]The culture method is basically the same as that of Example 2 for expressing VEGF humanized monoclonal antibodies, AGR2 humanized monoclonal antibodies, rabies antibodies, and resultsFigure 3-6The results showed that higher foreign protein expression can be obtained from commercial medium, thermostat group, and glucose group control group, which proves that combined sugars designed by the present invention are derived from commercial medium, thermostat group, and glucose group control group. Advanced technological effects have achieved excellent technical effects.

[0069]Table 8 Comparison of VEGF antibodies in different residents (unit: g / L)

[0070] CultureAntibody concentration Contrast 14.06 Contrast 24.77 Contrast 34.26 Contrast 44.18 Contrast 54.06 Contrast 63.65 Contrast 73.77 Contrast 83.89 Contrast 93.58 Contrast 104.01

[0071]Table 9 Comparison of AGR2 antibody expression of different feed medium (unit: g / L)

[0072] ...

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Abstract

A combined sugar feed medium for CHO cell culture and a medium addition method for CHO cells in view of temperature changes. During the culture process, the feed medium is added at different culture temperature nodes, and the temperature nodes are 30°C-34 in turn °C-37°C-34°C-32°C, the sugar concentration and combination of feed medium 1-5 are optimized, and the method can effectively increase the increase of exogenous protein.

Description

Technical field[0001]The present invention relates to the field of biotechnology, which is specifically located in the field of CHO cell culture medium.Background technique[0002]China hamster ovary cells (CHO) have been widely used in commercial production of therapeutic antibodies, with the continuous surge of the therapeutic antibody demand, the universality of CHO cells will continue to exist. Through animal cell culture, genetic engineering exogenous protein is a common method of obtaining a monoclonal antibody for treatment purposes, but since its expression is lower than the prokaryotic and yeast expression system, it is important to improve the expression efficiency of exogenous genes in CHO cells. Optimization medium is an important way to increase cellular activity and activate expression pathways.[0003]The current study of CHO cell-free-serum-free medium, the main method is to add protein hydrolyzate, insulin, transferrin, growth factor, vitamin, lipid, trace element, and ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10C12P21/08
CPCC07K16/10C07K16/18C07K16/22C07K16/2818C07K2317/14C07K2317/24C12N5/0682C12N2500/12C12N2500/14C12N2500/16C12N2500/20C12N2500/22C12N2500/32C12N2500/34C12N2500/36C12N2500/38C12N2501/33C12N2501/392C12N2510/02
Inventor 肖志华
Owner SHANGHAI OPM BIOSCI CO LTD