Repressing expression of difficult‐to‐express recombinant proteins during the selection process increases productivity of CHO stable pools

NRC publication: Yes

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Bibliographic Details
Published in:Biotechnology and bioengineering Vol. 120; no. 10; pp. 2840 - 2852
Main Authors: Maltais, Jean‐Sébastien, Lord Dufour, Simon, Morasse, Audrey, Stuible, Matthew, Loignon, Martin, Durocher, Yves
Format: Journal Article
Language:English
Published: United States Wiley 01.10.2023
Wiley Subscription Services, Inc
Subjects:
Antibodies
Bispecific antibodies
Cell surface receptors
Cell viability
Cellular stress response
CHO cells
cumate
Cytokines
difficult‐to‐express protein
Endoplasmic reticulum
ER stress
High mobility group proteins
HMGB1 protein
inducible expression
Metabolism
Monoclonal antibodies
produc
Productivity
Proteins
Receptors
ER stress
difficult-to-express protein
inducible expression
cumate
productivity
CHO cells
ISSN:1097-0290, 0006-3592, 1097-0290
Online Access:Get full text
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Summary:NRC publication: Yes
More than half of licensed therapeutic recombinant proteins (r‐proteins) are manufac-tured using constitutively‐expressing, stably‐transfected Chinese hamster ovary (CHO)clones. While constitutive CHO expression systems have proven their efficacy for themanufacturing of monoclonal antibodies, many next‐generation therapeutics such ascytokines and bispecific antibodies as well as biological targets such as ectodomains oftransmembrane receptors remain intrinsically challenging to produce. Herein, weexploited a cumate‐inducible CHO platform allowing reduced expression of variousclasses of r‐proteins during selection of stable pools. Following stable pool generation,fed‐batch productions showed that pools generated without cumate (OFF‐pools) weresignificantly more productive than pools selected in the presence of cumate (ON‐pools)for 8 out of the 10 r‐proteins tested, including cytokines, G‐protein coupled receptors(GPCRs), the HVEM membrane receptor ectodomain, the multifunctional protein HighMobility Group protein B1 (HMGB1), as well as monoclonal and bispecific T‐cell engagerantibodies. We showed that OFF‐pools contain a significantly larger proportion of cellsproducing high levels of r‐proteins and that these cells tend to proliferate faster whenexpression is turned off, suggesting that r‐protein overexpression imposes a metabolicburden on the cells. Cell viability was lower and pool recovery was delayed duringselection of ON‐pools (mimicking constitutive expression), suggesting that high producerswere likely lost or overgrown by faster‐growing, low‐producing cells. We also observed acorrelation between the expression levels of the GPCRs with Binding immunoglobulinProtein, an endoplasmic reticulum (ER) stress marker. Taken together, these data suggestthat using an inducible system to minimize r‐protein expression during stable CHO poolselection reduces cellular stresses, including ER stress and metabolic burden, leading topools with greater frequency of high‐expressing cells, resulting in improved volumetricproductivity.
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ISSN:1097-0290
0006-3592
1097-0290
DOI:10.1002/bit.28435