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Efficient serum-free culture medium

A serum-free medium and high-efficiency technology, applied in the direction of vertebrate cells, artificial cell constructs, animal cells, etc., can solve the problems of high cost and reduced expression, and achieve the effect of increasing growth, increasing expression, and reducing costs

Inactive Publication Date: 2015-01-21
SHANGHAI MBR BIOMEDICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The chemically defined medium containing recombinant proteins has disadvantages such as high cost. Removing recombinant proteins in the medium including insulin, IGF, HSA, and not reducing the expression level is the ultimate development direction of the medium in the future

Method used

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  • Efficient serum-free culture medium
  • Efficient serum-free culture medium
  • Efficient serum-free culture medium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1, preparation of serum-free medium

[0034] Table 1. Serum-free medium formulations

[0035]

[0036] Dissolve each component in pyrogen-free ultrapure water according to the formula in Table 1, and filter and sterilize to prepare the serum-free medium of the present invention.

Embodiment 2

[0037] Embodiment 2, optimizing glutamic acid, asparagine, the impact of aspartic acid concentration on cell growth

[0038] While maintaining the other nutrient components of the culture medium in accordance with Table 1, prepare culture media corresponding to different concentrations of glutamic acid, asparagine, and aspartic acid (Table 2). CHO cell lines expressing trastuzumab were treated according to 5x10 5 The concentration of cells / ml was inoculated, and cultured at 37 degrees, 110 rpm, and carbon dioxide concentration of 6.0%. When the glucose concentration was lower than 2 g / l, the sugar was added to 8 g / l. Cultured for 14 days, the number of cells reached the maximum on the 8th day, and Asp10mM / Asn10mM / Glu3mM produced the maximum cell density of 8.16x106 cells / ml. Asp6mM / Asn6mM / Glu6mM produced a cell density of 7.08x106 cells / ml (Table 2).

[0039]Table 2

[0040]

Embodiment 3

[0041] Example 3 Optimizing Glutamic Acid, Asparagine, and the Effect of Aspartic Acid Concentration on Cell Expression of Antibody

[0042] While maintaining the other nutrient components of the culture medium in accordance with Table 1, prepare culture media corresponding to different concentrations of glutamic acid, asparagine, and aspartic acid (Table 2). The CHO cell line expressing trastuzumab was inoculated at a concentration of 5x105 cells / ml, and cultured at 37 degrees, 110 rpm, and a carbon dioxide concentration of 6.0%. When the glucose concentration was lower than 2 g / L, supplement Sugar to 8 g / l. After culturing for 14 days, samples were taken on D14 to measure the expression level, wherein Asp6mM / Asn6mM / Glu6mM produced the highest expression level of 720 mg / L ( figure 1 ).

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Abstract

The invention belongs to the technical field of animal cell culture, and specifically relates to an efficient serum-free culture medium. The efficient serum-free culture medium disclosed by the invention mainly comprises amino acids, inorganic salts, a buffer system, vitamins, trace elements, carbon source and liquid substances. The culture medium provided by the invention may be similar to a commercially available culture medium in species of the components; the content of each component is obtained by long-term research; the content of each component is optimized; and each component is balanced. The efficient serum-free culture medium does not contain a culture medium system limited by chemical ingredients of the recombinant protein, can greatly promote the growth of CHO, increases expression amount, greatly reduces the cost of the culture medium, and is beneficial to process stability of the recombinant protein quality.

Description

technical field [0001] The invention belongs to the technical field of animal cell culture, and in particular relates to a serum-free culture medium. Background technique [0002] Culture medium refers to a liquid or solid medium that can maintain animal cells, microbial cells, plant cells, etc. growing in vitro. The field of this patent design is the liquid medium for culturing animal cells, especially CHO suspension cells. The culture medium currently used for animal cells can be divided into: medium containing serum, low serum medium, serum-free medium (medium containing certain components such as plant hydrolyzate), chemical medium according to the development process of animal culture medium. Defined media (chemically defined, but may contain some recombinant proteins) and chemically defined media without recombinant proteins, of which chemically defined media without recombinant proteins are the most advanced stage of media development . Genetically engineered CHO c...

Claims

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Application Information

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IPC IPC(8): C12N5/071
Inventor 陈亮都业杰
Owner SHANGHAI MBR BIOMEDICAL TECH
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