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Cell culture medium for improving antibody purity and cultivation method

A culture medium and feed medium technology, applied in chemical instruments and methods, biochemical equipment and methods, animal cells, etc., can solve problems such as increasing production costs and reducing antibody expression

Inactive Publication Date: 2016-12-14
SUNSHINE LAKE PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existing methods to improve the purity of antibodies are mainly to shorten the cell culture cycle in the upstream or sacrifice the yield in the downstream purification process. Shortening the cell culture cycle will reduce the expression of antibodies, and other glycosylation levels will also change; Although the method can obtain high-purity antibodies, it will increase the production cost

Method used

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  • Cell culture medium for improving antibody purity and cultivation method
  • Cell culture medium for improving antibody purity and cultivation method
  • Cell culture medium for improving antibody purity and cultivation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Basic medium: Hycell CHO Medium (purchased from Hyclone)

[0040] Self-made feed medium: Take 300mmol tyrosine (181.2g / mol), 280mmol cysteine ​​(121.15g / mol), 100mmol tryptophan (204.23g / mol), add under stirring and protected from light Into 700ml ultrapure water, stir for 40min, add NaOH (32.1g / L). Continue stirring for 30 minutes, adjust the pH to 10.95~11.2 with 5M NaOH, continue stirring for 20 minutes, determine the turbidity, the turbidity should be less than 10NTU, dilute to 1000ml, stir for more than 10 minutes, filter to a sterile reagent bottle with 0.22μm filter membrane.

[0041] Feeding medium: Acti CHO FeedACD (purchased from GE)

[0042] Cell line: CHO cell line containing nucleic acid encoding anti-human tumor necrosis factor (TNFα) antibody (refer to WO1997029131 patent)

[0043] Cultured in a 500ml shake flask, the cell line is 0.85×10 6 cells / mL for inoculation, initial culture volume 100ml, culture temperature 37℃, CO 2 The concentration is 8%, the rotation...

Embodiment 2

[0045] Basic medium: Hycell CHO Medium (purchased from Hyclone)

[0046] Self-made feed medium: Take 300mmol tyrosine (181.2g / mol), 210mmol cysteine ​​(121.15g / mol), 100mmol tryptophan (204.23g / mol), add under stirring and protected from light Into 700ml ultrapure water, stir for 40min, add NaOH (32.1g / L). Continue stirring for 30 minutes, adjust the pH to 10.95~11.2 with 5M NaOH, continue stirring for 20 minutes, determine the turbidity, the turbidity should be less than 10NTU, dilute to 1000ml, stir for more than 10 minutes, filter to a sterile reagent bottle with 0.22μm filter membrane.

[0047] Feeding medium: Acti CHO FeedACD (purchased from GE)

[0048] Cell line: CHO cell line containing nucleic acid encoding anti-human tumor necrosis factor (TNFα) antibody (refer to WO1997029131 patent)

[0049] Cultured in a 500ml shake flask, the cell line is 0.85×10 6 cells / mL for inoculation, initial culture volume 100ml, culture temperature 37℃, CO 2 The concentration is 8%, the rotation...

Embodiment 3

[0051] Basic medium: Hycell CHO Medium (purchased from Hyclone)

[0052] Self-made feed medium: Take 300mmol tyrosine (181.2g / mol), 140mmol cysteine ​​(121.15g / mol), 100mmol tryptophan (204.23g / mol), add under stirring and protected from light Into 700ml ultrapure water, stir for 40min, add NaOH (32.1g / L). Continue stirring for 30 minutes, adjust the pH to 10.95~11.2 with 5M NaOH, continue stirring for 20 minutes, determine the turbidity, the turbidity should be less than 10NTU, dilute to 1000ml, stir for more than 10 minutes, filter to a sterile reagent bottle with 0.22μm filter membrane.

[0053] Feeding medium: Acti CHO FeedACD (purchased from GE)

[0054] Cell line: CHO cell line containing nucleic acid encoding anti-human tumor necrosis factor (TNFα) antibody (refer to WO1997029131 patent)

[0055] Cultured in a 500ml shake flask, the cell line is 0.85×10 6 cells / mL for inoculation, initial culture volume 100ml, culture temperature 37℃, CO 2 The concentration is 8%, the rotation...

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Abstract

The invention relates to the field of biotechnology, in particular to a cell culture medium for improving antibody purity and a cultivation method, discloses a self-made feeding culture medium, and the culture medium contains 90mM to 500mM cysteine, and further provides a cell cultivation method. The cell cultivation method comprises the following steps: mammalian cells containing encoded antibody nucleic acid are inoculated into a basal culture medium; after the cells enter into a cellular proliferative stage, the self-made feeding culture medium is added; and cell density is monitored and incubation time is adjusted. By controlling the concentration of the cysteine in the self-made feeding culture medium, the adding moment and the adding quantity, the antibody purity can be improved remarkably, while the antibody purity is improved, the antibody expression quantity and normal glycosylation level can be maintained, and the antibody efficacy is ensured.

Description

Technical field [0001] The present invention relates to the field of biotechnology, in particular to a cell culture medium and culture method for improving antibody purity. Background technique [0002] In the biomedical industry, mammalian cells have obvious advantages as expression vectors for recombinant protein drugs. They can complete the complex post-translational modification of recombinant proteins and ensure the correct formation of complex protein structures such as disulfide bonds and glycosylation in the product. , So as to ensure the drug properties and efficacy of recombinant protein, it is an important type of expression system. [0003] In recent years, large-scale animal cell culture technology for monoclonal antibody production has developed rapidly. The technological progress in this field mainly focuses on the development of personalized culture media and optimization of process conditions. As a recombinant antibody of biological macromolecules, it has multipl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12P21/00
CPCC12N5/0602C07K16/2863C07K16/2866C12N2500/32
Inventor 程习文鄢成伟杨彬孙文正翁源灿邓崇飞
Owner SUNSHINE LAKE PHARM CO LTD
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