title: Chemical manipulation of the mTORC1 pathway in industrially relevant CHOK1 cells enhances production of therapeutic proteins.
abstract: The mammalian target of rapamycin complex 1 (mTORC1) is known as a central coordinator of protein synthesis and cell growth in response to the cellular environment. In this work, chemical manipulation of mTORC1 pathway was employed to enhance mAb production as well as increase understanding of intracellular pathways in GS-CHOK1 cells. Using the phosphorylation status of mTORC1 downstream targets, S6K1 and 4E-BP1, as read-outs of mTORC1 activity, we investigated the contribution of each target protein to growth and/or productivity. Inoculation of cultures in the presence of rapamycin, a specific inhibitor of mTORC1, increased viability and final titer. The initial increase in specific productivity and inhibition of growth by rapamycin correlated with diminished phospho-S6K1. However, inhibition was transient and cells recovered by unknown mechanisms. In contrast, phosphorylation of 4E-BP1 was preserved in response to rapamycin. Finally, we examined the activity of mTORC1 after addition of a custom-designed feed. Feeding led to substantial increase in growth and productivity and the phosphorylation of both targets was elevated. Though many details of mTORC1 signaling in CHO cells remain to be clarified, we have provided evidence that environmental manipulation of the mTORC1 pathway correlates with changes in cell growth and recombinant protein production.
authors.affiliation: Faculty of Life Sciences, University of Manchester, Manchester, UK. Nazanin.dadehbeigi@outlook.com.
authors.forename: Nazanin
authors.initials: N
authors.lastname: Dadehbeigi
authors.affiliation: Faculty of Life Sciences, University of Manchester, Manchester, UK.
authors.forename: Alan J
authors.initials: AJ
authors.lastname: Dickson
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics; D000818:Animals; D016466:CHO Cells; D018797:Cell Cycle Proteins; D049109:Cell Proliferation / Q000187:drug effects; D006224:Cricetinae; D003412:Cricetulus; D000076222:Mechanistic Target of Rapamycin Complex 1; D053858:Metabolic Networks and Pathways / Q000235:genetics*; D046912:Multiprotein Complexes / Q000737:chemistry / Q000235:genetics*; D010750:Phosphoproteins / Q000235:genetics; D010766:Phosphorylation; D014176:Protein Biosynthesis / Q000187:drug effects; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D020123:Sirolimus / Q000494:pharmacology; D058570:TOR Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; C087000:EIF4EBP1 protein, human; D046912:Multiprotein Complexes; D010750:Phosphoproteins; D011994:Recombinant Proteins; D000076222:Mechanistic Target of Rapamycin Complex 1; D058570:TOR Serine-Threonine Kinases; D020123:Sirolimus
keywords: CHOK1; Eukaryotic initiation factor 4E binding protein 1 (4E-BP1); Rapamycin; mAb production; mTOR Complex 1