Abstract

Capitalizing on the ability of mammalian cells to conduct complex post-translational modifications, most protein therapeutics are currently produced in cell culture systems. Addition of a signal peptide to the product protein enables its accumulation in the cell culture supernatant, but separation of the product from endogenously secreted proteins remains costly and labor-intensive. We considered that global downregulation of translation of non-product proteins would be an efficient strategy to minimize downstream processing requirements. Therefore, taking advantage of the ability of mammalian protein kinase R (PKR) to switch off most cellular translation processes in response to infection by viruses, we fused a caffeine-inducible dimerization domain to the catalytic domain of PKR. Addition of caffeine to this construct results in homodimerization and activation of PKR, effectively rewiring rapid global translational downregulation to the addition of the stimulus in a dose-dependent manner. Then, to protect translation of the target therapeutic, we screened viral and cellular internal ribosomal entry sites (IRESes) known or suspected to be resistant to PKR-induced translational stress. After choosing the best-in-class Seneca valley virus (SVV) IRES, we additionally screened for IRES transactivation factors (ITAFs) as well as for supplementary small molecules to further boost the production titer of the product protein under conditions of global translational downregulation. Importantly, the residual global translation activity of roughly 10% under maximal downregulation is sufficient to maintain cellular viability during a production timeframe of at least five days. Standard industrially used adherent as well as suspension-adapted cell lines transfected with this synthetic biology-inspired Protein Kinase R-Enhanced Protein Production (PREPP) system could produce several medicinally relevant protein therapeutics, such as the blockbuster drug rituximab, in substantial quantities and with significantly higher purity than previous culture technologies. We believe incorporation of such purity-by-design technology in the production process will alleviate downstream processing bottlenecks in future biopharmaceutical manufacturing.