Related Experiment Video
Updated: Apr 26, 2026
A High-throughput Automated Platform for the Development of Manufacturing Cell Lines for Protein Therapeutics
Published on: September 22, 2011
It's time to regulate: coping with product-induced nongenetic clonal instability in CHO cell lines via regulated
Shahram Misaghi1, Jennifer Chang, Brad Snedecor
1Dept. of Early Stage Cell Culture, Genentech, Inc, 1 DNA way, South San, Francisco, CA, 94080.
Abstract:
Clonal instability and titer loss during Chinese hamster ovary (CHO) cell line development (CLD) has several underlying causes, the most prominent of which are DNA copy number loss and DNA silencing. However, in some cases, clonal instability is due to the toxicity of the therapeutic protein(s) that clones express. Unlike DNA copy number loss, which may occur in some clones or DNA silencing that is prevalent in certain regions of the genome, the hallmark of product induced clonal instability is its manifestation in all the selected clones. To circumvent such product induced clonal instability, we have developed a vector construct that utilizes a regulated protein expression system in which the constitutive expression of the target protein(s) is prevented unless doxycycline is added to the culture. We have then successfully used this system to express, at high titers, an antibody for which constitutive expression results in clonal instability perhaps due to intracellular accumulation of the antibody. Our data shows that unlike the constitutively expressed or continuously induced clones, uninduced clones do not display instability. Furthermore, maintaining the uninduced clones in culture for months or subjecting them to freeze-thaws did not have any effects on their titers. All together, our findings suggest that a regulated expression system could be suitable for production of difficult proteins that trigger instability.
Insights
Product-induced clonal instability in Chinese hamster ovary (CHO) cell line development can be overcome using a regulated expression system. This doxycycline-inducible system prevents protein toxicity, ensuring stable high-titer antibody production.
Area of Science:
- Biotechnology
- Cell Line Development
- Protein Expression
Background:
- Clonal instability and titer loss in Chinese hamster ovary (CHO) cell line development (CLD) are often caused by DNA copy number loss or silencing.
- Product-induced clonal instability, stemming from therapeutic protein toxicity, affects all selected clones, unlike genetic instability factors.
Purpose of the Study:
- To develop and validate a regulated protein expression system to circumvent product-induced clonal instability in CHO cell lines.
- To enable stable, high-titer production of therapeutic proteins that cause instability when constitutively expressed.
Main Methods:
- Engineered a novel vector construct utilizing a doxycycline-regulated protein expression system.
- Prevented constitutive expression of target proteins until doxycycline induction.
- Cultured and evaluated clones under induced and uninduced conditions, including long-term culture and freeze-thaw cycles.
Main Results:
- The regulated expression system successfully produced a therapeutic antibody at high titers.
- Uninduced clones exhibited no instability, maintaining consistent titers over extended periods and after stress.
- Induced or constitutively expressed clones displayed instability, confirming the system's efficacy.
Conclusions:
- A regulated expression system is effective in preventing product-induced clonal instability in CHO cell line development.
- This approach is suitable for the stable production of difficult-to-express therapeutic proteins that trigger instability.
- The doxycycline-inducible system offers a robust solution for maintaining high protein titers and clonal stability.
More Related Videos
Related Concept Videos
02:39Positive Regulator Molecules
01:23Negative Regulator Molecules
01:16Cell Lines
01:28The Cell Cycle Control System
Cyclins and cyclin-dependent kinases (Cdks) are the primary cell cycle regulators and...