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Mammalian cell culture processes for protein production

A cell culture and cell technology, applied in the field of new process of culturing mammalian cells, can solve the unreported problems of dextran sulfate or polyvinyl sulfate effect, etc., to achieve increased titer, high cell viability, and extended production period Effect

Active Publication Date: 2015-08-05
BRISTOL MYERS SQUIBB CO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the effect of dextran sulfate or polyvinyl sulfate during the death phase has not been reported

Method used

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  • Mammalian cell culture processes for protein production
  • Mammalian cell culture processes for protein production
  • Mammalian cell culture processes for protein production

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 3

[0040] The fed-batch culture production assay described in Example 3 found that conditions without GF increased the peak viable cell density from 13.0 × 10 6 cells / ml increased to 15.7 × 10 6 cells / ml, and improved cell viability at later stages of culture, from 59.4% to 96.2% in the GF condition on day 8, and from 35.6% to 65.1% on day 14. As a result, the protein titer increased by 80.9%. Furthermore, protein quality was enhanced under GF-free conditions, as macromolecular species decreased from 14.3% to 4.8% under GF conditions (see Figure 4 ).

[0041] also, Figure 5 – 8 shows that the generation stability of cells is improved in the absence of GF in three different clones. After culturing cells under GF conditions, productivity decreased, whereas cells cultured without GF maintained their productivity.

[0042] Overall, our results demonstrate that chemically defined media free of proteins and peptides are able to propagate CHO cells, deliver comparable productivi...

Embodiment 1

[0125] CHO cells are able to grow without GF

[0126] 1. Thaw a new vial of cells from the previous passage and grow in platform medium (containing 1 or 10 mg / L insulin) for 2 passages.

[0127] 2. At passage 3, transfer the cells to basal medium without insulin and inject with 0.6x10 6 The density of cells / ml was kept splitting.

[0128] 3. In the first few passages under insulin-free conditions, cell growth can be significantly slowed and viability can be reduced (~90%). At the same time, ammonium production can be increased due to insufficient glucose uptake and oxidation of amino acids as energy sources. As a result, the pH in the flask can increase. CO 2 Levels may need to be adjusted / increased accordingly in order to control the pH in the range of 7.0-7.3. This is very important for maintaining cells in a healthy state.

[0129] 4. In general, less than 30% of the spent medium maintained to new passages should be maintained. In cases where cells are growing too sl...

Embodiment 2

[0134] Removing insulin did not drastically affect the mTOR pathway

[0135] The purpose of this study was to use antibody arrays to compare the phosphorylation / expression levels of proteins involved in mTOR in cells grown with and without the growth factor insulin, thereby demonstrating that the cells are able to function in Grow without GF.

[0136] Antibody Array Sample Preparation

[0137] Clone A was grown on basal medium without or with insulin. On day 3, sample 5x 10 for each condition 6living cells. Cells were centrifuged down at 500 g for 5 min at 4°C. Cells were washed with 10 ml ice-cold 1xPBS and centrifuged down at 500g for 5 min at 4°C. Keep cells on ice or at 4 °C throughout sample processing. Immediately after pouring the PBS, the cells were frozen at -70°C and stored at -70°C until antibody array analysis.

[0138] Antibody Array Protocol

[0139] protein extraction

[0140] Wash cells with ice-cold 1X PBS. Lysis beads and extraction buffer are added...

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Abstract

The present invention describes methods and processes for the production of proteins by animal cell or mammalian cell culture. In one aspect, the methods comprise the growth of cells in a growth factor / protein / peptide free medium. In another aspect, the methods comprise the addition of growth factors during the production phase. The methods sustain a high viability of the cultured cells, and can yield an increased end titer of protein product, and a high quality of protein product.

Description

field of invention [0001] The present invention relates to a novel process for culturing mammalian cells that produce protein products. The performance of the cell culture process results in high cell viability and / or cell density and may also result in high product quality and productivity. Background of the invention [0002] Preference is given to using animal cell cultures, especially mammalian cell cultures, for expression of recombinantly produced proteins for therapeutic and / or prophylactic applications. [0003] In general, protein expression levels in mammalian cell culture-based systems are significantly lower than in microbial expression systems (eg, bacterial or yeast expression systems). However, bacterial and yeast cells are limited in their ability to optimally express high-molecular-weight protein products, correctly fold proteins with complex three-dimensional structures, and / or provide post-translational modifications necessary for maturation of expressed ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/00C12N15/62C12N15/63C12P21/02
CPCC12P21/02C07K16/2875C12N2501/33C07K2317/569C07K2319/30C07K14/70521C12N5/005C12N2510/02
Inventor 田军M.博里斯李正剑N.阿布亚布西区玉翠钱南新戴晓萍
Owner BRISTOL MYERS SQUIBB CO
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