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Reducing recombinant protein expression during CHO pool selection enhances frequency of high-producing cells
Adeline Poulain1, Alaka Mullick2, Bernard Massie1
1National Research Council Canada, 6100 Royalmount Avenue, Montréal, QC H4P 2R2, Canada; Département de microbiologie et immunologie, Faculté de médecine, Université de Montréal, QC, Canada.
Journal of Biotechnology
|March 20, 2019
Summary
Using an inducible expression system, researchers found that down-regulating protein production during Chinese hamster ovary (CHO) cell selection significantly increases the frequency of high-producing clones for biologics manufacturing.
Area of Science:
- Biotechnology
- Cell Biology
- Bioprocessing
Background:
- Chinese hamster ovary (CHO) cells are crucial for industrial production of biologics like monoclonal antibodies (mAbs).
- Identifying high-producing CHO cell lines is challenging due to metabolic burden and stress from high recombinant protein (r-protein) expression.
- Traditional constitutive expression systems offer limited optimization for mitigating these selection stresses.
Purpose of the Study:
- To evaluate the impact of an inducible expression system on selecting high-producing CHO cell clones.
- To compare the frequency of high-producer clones using an inducible system versus a constitutive system during selection.
- To assess the effects of r-protein overexpression on cell recovery, stress markers, and viability during selection.
Main Methods:
- Development of a CHO cell line (CHO BRI/rcTA) with a cumate gene switch for inducible r-protein expression.
- Generation of transfected cell pools with the cumate gene switch in either an "on-state" (high expression) or "off-state" (down-regulated expression) during selection.
- Culturing cells in fed-batch processes and analyzing volumetric productivity, pool recovery, BiP expression, and cell death.
Main Results:
- Down-regulating r-protein expression ("off-state") during pool selection significantly enhanced the frequency of high-producing stable clones compared to maintaining high expression ("on-state").
- The "on-state" selection led to reduced pool recovery, increased BiP expression (an ER stress marker), and higher cell death.
- Inducible CHO BRI/rcTA pools achieved high volumetric productivity (up to 1.1 g/L) within two weeks of transfection.
Conclusions:
- Regulating r-protein expression during pool generation provides a substantial advantage for selecting high-producing CHO cell clones.
- The cumate gene switch effectively mitigates metabolic burden and stress during clone selection, improving efficiency.
- Inducible expression systems are valuable for scalable biologics production in CHO cells, enhancing stable clone generation.
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