Most therapeutic monoclonal antibodies in biopharmaceutical processes are produced in Chinese hamster ovary (CHO) cells. Technological advances have rendered the selection procedure for higher producers a robust protocol. However, information on molecular mechanisms that impart the property of hyper-productivity in the final selected clones is currently lacking. In this study, an IgG-producing industrial cell line and its methotrexate (MTX)-amplified progeny cell line were analyzed using transcriptomic, proteomic, phosphoproteomic, and chromatin immunoprecipitation (ChIP) techniques. Computational prediction of transcription factor binding to the transgene cytomegalovirus (CMV) promoter by the Transcription Element Search System and upstream regulator analysis of the differential transcriptomic data suggested increased in vivo CMV promoter-cAMP response element binding protein (CREB1) interaction in the higher producing cell line. Differential nuclear proteomic analysis detected 1.3-fold less CREB1 in the nucleus of the high productivity cell line compared with the parental cell line. However, the differential abundance of multiple CREB1 phosphopeptides suggested an increase in CREB1 activity in the higher producing cell line, which was confirmed by increased association of the CMV promotor with CREB1 in the high producer cell line. Thus, we show here that the nuclear proteome and phosphoproteome have an important role in regulating final productivity of recombinant proteins from CHO cells, and that CREB1 may play a role in transcriptional enhancement. Moreover, CREB1 phosphosites may be potential targets for cell engineering for increased productivity.

Viral contamination in biopharmaceutical manufacturing can lead to shortages in the supply of critical therapeutics. To facilitate the protection of bioprocesses, we explored the basis for the susceptibility of CHO cells to RNA virus infection. Upon infection with certain ssRNA and dsRNA viruses, CHO cells fail to generate a significant interferon (IFN) response. Nonetheless, the downstream machinery for generating IFN responses and its antiviral activity is intact in these cells: treatment of cells with exogenously-added type I IFN or poly I:C prior to infection limited the cytopathic effect from Vesicular stomatitis virus (VSV), Encephalomyocarditis virus (EMCV), and Reovirus-3 virus (Reo-3) in a STAT1-dependent manner. To harness the intrinsic antiviral mechanism, we used RNA-Seq to identify two upstream repressors of STAT1: Gfi1 and Trim24. By knocking out these genes, the engineered CHO cells exhibited activation of cellular immune responses and increased resistance to the RNA viruses tested. Thus, omics-guided engineering of mammalian cell culture can be deployed to increase safety in biotherapeutic protein production among many other biomedical applications.