Optimized cell culture medium and cell culture method, and application of optimized cell culture medium and cell culture method to preparation of protein and antibody

A medium and basic medium technology, applied in artificial cell constructs, cells modified by introducing foreign genetic material, animal cells, etc., can solve the problems of easy depletion of glutamine and accumulation of ammonium ions, and achieve high sugar The effect of the level of methylation and biological activity, simple production process, and stable protein properties

Active Publication Date: 2015-09-09
CHENGDU JINKAI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In summary, the existing technology has the key technical problem that glutamine is easily depleted and its metabolic by-product ammonium ions are seriously accumulated in the process of animal cell culture.

Method used

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  • Optimized cell culture medium and cell culture method, and application of optimized cell culture medium and cell culture method to preparation of protein and antibody
  • Optimized cell culture medium and cell culture method, and application of optimized cell culture medium and cell culture method to preparation of protein and antibody
  • Optimized cell culture medium and cell culture method, and application of optimized cell culture medium and cell culture method to preparation of protein and antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Embodiment 1. Preparation of culture medium

[0060] According to the formulations in Table 1 and Table 2, glutamic acid and 4mM glutamine of different concentrations were added to the basal medium as an optimized production medium in the culture process of recombinant CHO engineered cell lines, and only supplemented with the basal medium The comparison of glutamine control medium is used for the production and preparation of therapeutic target proteins under the same cell culture process conditions, so as to evaluate the effects of the two on the ammonium ion level of metabolic by-products, cell density, cell viability, and target protein glycosyl The influence of chemical level and biological activity, etc.

[0061] Table 1

[0062] Medium number

basal medium

Addition 1

Addition 2

A

optimized medium

EX-CELL302

Glutamate 2mM

Glutamine 4mM

B

control medium

EX-CELL302

Glutamine 4mM

C

o...

Embodiment 2

[0068] Example 2 Recombinant CHO Engineering Cell Culture Expressing Recombinant Human Type II Tumor Necrosis Factor Receptor-Antibody (rhTNFRII-Fc) Fusion Protein

[0069] 1. Construction of rhTNFRII-Fc fusion protein expression vector

[0070] The dhfr (dihydrofolate reductase) expression unit in the pSV2-dhfr vector (ATCC product) was cloned into the pCDNA3.1(+) vector (Invitrogen company product) by PCR technology and DNA recombination technology to construct a mammal expressing DHFR Animal cell expression vector pBF01.

[0071] According to the gene sequence of hTNFRII (Genebank Accession No. AH006638) and human IgG1 Fc fragment gene sequence (WO9411026), primers were designed for PCR amplification and cloned into the pUC18 plasmid.

[0072] The primers were redesigned, and the cDNA fragments of hTNFRII and human IgG1 Fc obtained above were overlapped by PCR amplification to obtain the full-length cDNA fragment of the rhTNFRII-Fc fusion protein, which was cloned into the...

Embodiment 3

[0086] Example 3. Preparation of recombinant human type II tumor necrosis factor receptor-antibody (rhTNFRII-Fc) fusion protein

[0087] 1. Separation and purification of rhTNFRII-Fc fusion protein:

[0088] The cell culture fluid obtained in Example 2 was sequentially filtered through DOHC and B1HC depth filters of Millipore Company, and the filtrate was collected and purified by Protein A affinity chromatography.

[0089] After the MabselectSuRe (GE Company) chromatographic column was washed and balanced with PBS buffer, the cell culture supernatant was loaded at a speed of 50-300 cm / h, and washed with PBS buffer until all unbound proteins were eluted (with A280 was monitored), then the fusion protein was eluted with 20mmol / L citric acid (pH3.5), and the fusion protein stock solution was collected to adjust the pH value to 7.2 with Tris.

[0090] 2. Detection and analysis

[0091] (1) Determination of sialic acid content in rhTNFRII-Fc fusion protein

[0092] According to...

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Abstract

The invention provides an optimized serum-free cell culture medium which is composed of a basal culture medium and additives, wherein the additives comprise glutamic acid with the final concentration of 2-5 mM and glutamine with the final concentration of 4 mM. The invention further provides the application of the cell culture medium to the culture of mammalian cells for production of therapeutic recombinant protein such as II type tumor necrosis factor receptor-antibody fusion protein (rhTNFRII-Fc)) and/or antibody such as CD20 monoclonal antibody drugs. By using the optimized cell culture medium for culturing mammalian cells, influences of metabolic by-products such as ammonium ions generated in the cell production culture process on the mammalian cell growing status and functions can be reduced and properties and yields of recombinant protein and the antibody are ensured.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular, the invention relates to a cell culture medium, a cell culture process and its application in the preparation of proteins and antibodies. Background technique [0002] Mammalian cells can accurately complete a series of post-translational modification processes such as glycosylation, disulfide bond folding, and phosphorylation, and the expressed protein products are closest to natural proteins or antibodies in terms of molecular structure and biological characteristics. Animal cells have been widely used for the expression of protein drugs. Large-scale mammalian cell culture has become a widely used technical method in biological and medical research and application, and the use of animal cell culture to produce enzymes, growth factors, vaccines and monoclonal antibodies with important medical value has become a key step in the high-tech industry of medicine and biology. important par...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N5/10C12P21/02C12P21/08
Inventor 钟绍东吴浪波唐德芳
Owner CHENGDU JINKAI BIOTECH CO LTD
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