Repressing expression of difficult-to-express recombinant proteins during the selection process increases productivity of CHO stable pools.
CHO cells
ER stress
cumate
difficult-to-express protein
inducible expression
productivity
Journal
Biotechnology and bioengineering
ISSN: 1097-0290
Titre abrégé: Biotechnol Bioeng
Pays: United States
ID NLM: 7502021
Informations de publication
Date de publication:
10 2023
10 2023
Historique:
revised:
04
05
2023
received:
11
01
2023
accepted:
09
05
2023
medline:
15
9
2023
pubmed:
26
5
2023
entrez:
26
5
2023
Statut:
ppublish
Résumé
More than half of licensed therapeutic recombinant proteins (r-proteins) are manufactured using constitutively-expressing, stably-transfected Chinese hamster ovary (CHO) clones. While constitutive CHO expression systems have proven their efficacy for the manufacturing of monoclonal antibodies, many next-generation therapeutics such as cytokines and bispecific antibodies as well as biological targets such as ectodomains of transmembrane receptors remain intrinsically challenging to produce. Herein, we exploited a cumate-inducible CHO platform allowing reduced expression of various classes of r-proteins during selection of stable pools. Following stable pool generation, fed-batch productions showed that pools generated without cumate (OFF-pools) were significantly 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 High Mobility Group protein B1 (HMGB1), as well as monoclonal and bispecific T-cell engager antibodies. We showed that OFF-pools contain a significantly larger proportion of cells producing high levels of r-proteins and that these cells tend to proliferate faster when expression is turned off, suggesting that r-protein overexpression imposes a metabolic burden on the cells. Cell viability was lower and pool recovery was delayed during selection of ON-pools (mimicking constitutive expression), suggesting that high producers were likely lost or overgrown by faster-growing, low-producing cells. We also observed a correlation between the expression levels of the GPCRs with Binding immunoglobulin Protein, an endoplasmic reticulum (ER) stress marker. Taken together, these data suggest that using an inducible system to minimize r-protein expression during stable CHO pool selection reduces cellular stresses, including ER stress and metabolic burden, leading to pools with greater frequency of high-expressing cells, resulting in improved volumetric productivity.
Substances chimiques
Recombinant Proteins
0
Antibodies, Monoclonal
0
Cytokines
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
2840-2852Informations de copyright
© 2023 National Research Council Canada and The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC. Reproduced with the permission of the Minister of Innovation, Science, and Economic Development.
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