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

Abstract

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.