Enhancing therapeutic protein production in recombinant CHO cell lines using small molecule epigenetic modulators

Abstract

For decades, the growing demands for therapeutic proteins have been emphasized the importance of enhancing the productivity of Chinese hamster ovary (CHO) cells. The addition of small molecules during recombinant CHO (rCHO) cell culture is a straightforward and effective strategy to achieve high levels of therapeutic protein production. Notably, small molecule epigenetic modulators like histone deacetylase inhibitor (iHDAC) and DNA methyltransferase inhibitor (iDNMT) have shown their potential in elevating therapeutic protein production in rCHO cell cultures. However, while the addition of a small number of iHDACs and iDNMTs is known to enhance productivity, the precise mechanisms remained incompletely elucidated, and they have shown drawbacks, such as a decrease of viable cell concentration. Thus, this study aims to identify novel efficient small molecule epigenetic modulators and elucidate their mechanisms in rCHO cell cultures. Additionally, to alleviate the decrease of viable cell concentration, a CRISPR/Cas9 knockout (KO) cell library was utilized for screening in addition of iHDAC condition and identified novel KO target genes. Firstly, to identify small molecule epigenetic modulators that enhance recombinant protein expression in CHO cells, I examined eight histone deacetylase inhibitors (iHDACs) and six DNA methyltransferase inhibitors as chemical additives in rCHO cell cultures. Among these, a benzamide-based iHDAC, CI994, was the most effective in increasing monoclonal antibody (mAb) production. Despite suppressing cell growth, the addition of CI994 to mAb-expressing GSR cell cultures at $10 \mu M$ resulted in a 2.3-fold increase in maximum mAb concentration due to a 3.0-fold increase in specific mAb productivity ($q_P$). CI994 increased mRNA levels of mAb and histone H3 acetylation in GSR cells. In addition, 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 $q_P$. Similar beneficial effects of CI994 on mAb production were observed in other benzamide-based iHDACs (MS-275, Mocetinostat, Tucidinostat). Collectively, the findings indicate that benzamide-based iHDACs increases mAb production in rCHO cell cultures by chromatin remodeling resulting from acetylation of histones in the mAb gene promoter. However, the addition of iHDACs has been found to decrease viable cell concentration in rCHO cell culture, thereby offsetting the potential enhancement of therapeutic protein concentration compared to enhanced qP. Thus, secondly, to mitigate the iHDAC-induced negative effect on viable cell concentration, a screening using a genome-wide CRISPR/Cas9 gene KO cell library in rCHO cells was performed under CI994 and identified 10 potential genes that could enhance viable cell concentration of rCHO cell cultures under CI994 treatment. Among these genes, Bcor was validated as a promising target for improving viable cell concentration without negatively affecting rCHO cell productivity in iHDAC-containing conditions. The KO of Bcor resulted in increased viable cell concentration as well as therapeutic protein concentration. These findings highlight the potential of screening using a gene KO cell library to identify relevant target genes for overcoming challenges in rCHO cell cultures. Particularly, the knockout of Bcor, as identified in this study, showcased its applicability in achieving higher levels of therapeutic protein productivity under addition of iHDAC conditions. In conclusion, this study successfully identified novel small molecule epigenetic modulators that improve the productivity of therapeutic protein production in rCHO cell lines, and elucidated the underlying mechanism of productivity improvement of these small molecules. In addition, through screening with a genome-wide CRISPR/Cas9-based gene KO cell library, potential target genes were identified to mitigate the decrease in viable cell concentration induced by small molecule epigenetic modulators. Finally, through the KO of the gene Bcor, which was one of the genes identified through screening, a higher viable cell concentration was achieved compared to the control cells and also increase of therapeutic protein concentration was observed.