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Updated: May 12, 2026
Rapid Antibody Glycoengineering in Chinese Hamster Ovary Cells
Published on: June 2, 2022
Resilient immortals, characterizing and utilizing Bax/Bak deficient Chinese hamster ovary (CHO) cells for high titer
Shahram Misaghi1, Yan Qu, Andrew Snowden
1Dept. of Early Stage Cell Culture, Genentech, Inc., 1 DNA way, South San Francisco, CA, 94080, USA.
Abstract:
Cell death due to apoptosis is frequently observed in large-scale manufacturing of therapeutic proteins, and can reduce product accumulation in bioreactors. Several different strategies that involve overexpression of antiapoptotic or downregulation of proapoptotic proteins have been designed in attempt to curb this problem in Chinese hamster ovary (CHO) cell culture. However, each of these designs has their own shortcomings and limits, rendering them ineffective for large-scale protein production. Recently, we have reported generation of a Bax and Bak deficient dhfr(-/-) CHO cell line using zinc-finger nucleases. Here we demonstrate that puromycin, but not methotrexate, selection can be used to generate antibody-expressing Bax and Bak deficient clones that are not only resistant to apoptosis, but that can also achieve higher titers relative to parental CHO cells due to higher cell density. Additionally, we show that Bax and Bak deficient cells have more mitochondria with healthy membrane potential, an attribute that perhaps contributes to their more potent growth compared to parental cells. Bax and Bak deficient cells do not readily apoptose, as shown by the ability to withstand high concentrations of apoptosis inducing agents, such as sodium butyrate, without a reduction in viability, growth, or titer. These traits render Bax and Bak deficient cells a potentially attractive host for production of therapeutic proteins at industrial scale.
Insights
Engineering Chinese hamster ovary (CHO) cells deficient in Bax and Bak proteins enhances therapeutic protein production by increasing cell density and resistance to apoptosis. This improved cell line offers a promising host for industrial-scale biomanufacturing.
Area of Science:
- Biotechnology
- Cell Biology
- Bioprocessing
Background:
- Apoptosis, or programmed cell death, is a significant challenge in large-scale therapeutic protein manufacturing using Chinese hamster ovary (CHO) cells, leading to reduced product yields.
- Existing strategies to mitigate apoptosis, such as manipulating antiapoptotic or proapoptotic proteins, have limitations for industrial applications.
Purpose of the Study:
- To evaluate the efficacy of Bax and Bak deficient CHO cells for enhanced therapeutic protein production.
- To assess the apoptosis resistance and growth characteristics of these engineered cells.
Main Methods:
- Generation of a Bax and Bak deficient dhfr(-/-) CHO cell line using zinc-finger nucleases.
- Selection of antibody-expressing clones using puromycin.
- Assessment of apoptosis resistance, cell density, mitochondrial health, and protein titers.
Main Results:
- Puromycin selection successfully generated apoptosis-resistant, antibody-expressing Bax and Bak deficient CHO clones.
- These clones exhibited higher cell densities and consequently achieved higher therapeutic protein titers compared to parental CHO cells.
- Bax and Bak deficient cells demonstrated enhanced mitochondrial membrane potential and greater resistance to apoptosis-inducing agents like sodium butyrate.
Conclusions:
- Bax and Bak deficient CHO cells are robust hosts for therapeutic protein production, showing resistance to apoptosis and improved growth.
- These engineered cells represent a valuable platform for optimizing industrial-scale biomanufacturing of therapeutic proteins.
