Small molecule epigenetic modulators that modify epigenetic states in cells are useful tools for regulating gene expression by inducing chromatin remodeling. To identify small molecule epigenetic modulators that enhance recombinant protein expression in Chinese hamster ovary (CHO) cells, we examined eight histone deacetylase inhibitors (iHDACs) and six DNA methyltransferase inhibitors as chemical additives in recombinant CHO (rCHO) cell cultures. Among these, a benzamidebased iHDAC, CI994, was the most effective in increasing monoclonal antibody (mAb) production. Despite suppressing cell growth, the addition of CI994 to mAbexpressing GSR cell cultures at 10 μM resulted in a 2.3-fold increase in maximum mAb concentration due to a 3.0-fold increase in specific mAb productivity (q mAb ).CI994 increased mAb messenger RNA levels and histone H3 acetylation in GSR cells, and chromatin immunoprecipitation-quantitative polymerase chain reaction analysis revealed that CI994 significantly increased the histone H3 acetylation level at the cytomegalovirus promoter driving mAb gene expression, indicating that chromatin remodeling in the promoter region results in enhanced mAb gene transcription and q mAb . Similar beneficial effects of CI994 on mAb production were observed in mAbexpressing CS13-1.00 cells. Collectively, our findings indicate that CI994 increases mAb production in rCHO cell cultures by chromatin remodeling resulting from acetylation of histones in the mAb gene promoter.

Cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger essential to many biological processes. Forskolin, a labdane diterpene, is widely used to increase cAMP levels in various cell types. When 5 µm of forskolin is added to recombinant Chinese hamster ovary (rCHO) cell cultures producing monoclonal antibody (mAb) (GSR cell line), it decreases specific growth rate (), but increases culture longevity and specific mAb productivity (q mAb). The beneficial effect of forskolin on culture longevity and q mAb outweighs its detrimental effect on , resulting in 1.5-fold increase in the maximum mAb concentration (MMC) without an adverse effect on mAb quality attributes such as aggregation, charge variation, and galactosylation. Forskolin induces cell cycle arrest at the G0/G1 phase via the LKB1/AMPK pathway and inhibits apoptosis via the CREB/Bcl-2 pathway. The beneficial effect of forskolin on mAb production is also demonstrated with another rCHO cell line (ABTAA). Addition of 5 µm forskolin to ABTAA cell cultures also results in 1.5-fold increase in the MMC. Taken together, the results obtained demonstrate that forskolin is an efficient chemical reagent to increase mAb production in rCHO cell cultures.