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.

Due to the high medical and commercial value of recombinant proteins for clinical and diagnostic purposes, the protein synthesis machinery of mammalian host cells is the subject of extensive research by the biopharmaceutical industry. RNA translation and protein synthesis are steps that may determine the extent of growth and productivity of host cells. To address the problems of utilization of current radioisotope methods with proprietary media, we have focused on the application of an alternative method of measuring protein synthesis in recombinant Chinese hamster ovary (CHO) cells. This method employs puromycin as a nonradioactive label which incorporates into nascent polypeptide chains and is detectable by western blotting. This method, which is referred to as SUnSET, successfully demonstrated the expected changes in protein synthesis in conditions that inhibit and restore translation activity and was reproducibly quantifiable. The study of the effects of feed and sodium butyrate addition on protein synthesis by SUnSET revealed an increase following 1 h feed supplementation while a high concentration of sodium butyrate was able to decrease translation during the same treatment period. Finally, SUnSET was used to compare protein synthesis activity during batch culture of the CHO cell line in relation to growth. The results indicate that as the cells approached the end of batch culture, the global rate of protein synthesis declined in parallel with the decreasing growth rate. In conclusion, this method can be used as a "snapshot" to directly monitor the effects of different culture conditions and treatments on translation in recombinant host cells.

Recombinant human Factor IX (rFIX) was cloned in a mammalian expression vector and transfected into CHO and HEK-293. Treatment with 10(-9) M methyl testosterone increased rFIX production by 30-50% in CHO and HEK clones. However, 10(-9) M 17beta-oestradiol increased production of rFIX by ~50% in CHO-F7 clone and decreased production by 48% and 37% in CHO-F8 and HEK-F2-6, respectively. Progesterone treatment inhibited rFIX production in both cell lines. Production of rFIX can thus be increased by sex hormone treatment and therefore used to enhance biotechnological production in mammalian cells.