Overexpression of transcription factor Foxa1 and target genes remediate therapeutic protein production bottlenecks in Chinese hamster ovary cells

Abstract

Despite extensive research conducted to increase protein production from Chinese hamster ovary (CHO) cells, cellular bottlenecks often remain, hindering high yields. In this study, a transcriptomic analysis led to the identification of 32 genes that are consistently upregulated in high producer clones and thus might mediate high productivity. Candidate genes were associated with functions such as signaling, protein folding, cytoskeleton organization, and cell survival. We focused on two engineering targets, Erp27, which binds unfolded proteins and the Erp57 disulfide isomerase in the endoplasmic reticulum, and Foxa1, a pioneering transcription factor involved in organ development. Erp27 moderate overexpression increased production of an easy‐to‐express antibody, whereas Erp27 and Erp57 co‐overexpression increased cell density, viability, and the yield of difficult‐to‐express proteins. Foxa1 overexpression increased cell density, cell viability, and easy‐ and difficult‐to‐express protein yields, whereas it decreased reactive oxygen species late in fed‐batch cultures. Foxa1 overexpression upregulated two other candidate genes that increased the production of difficult‐ and/or easy‐to‐express proteins, namely Ca3, involved in protecting cells from oxidative stress, and Tagap, involved in signaling and cytoskeleton remodeling. Overall, several genes allowing to overcome CHO cell bottlenecks were identified, including Foxa1, which mediated multiple favorable metabolic changes that improve therapeutic protein yields.

Methylthioadenosine (MTA) boosts cell‐specific productivities of Chinese hamster ovary cultures: dosage effects on proliferation, cell cycle and gene expression

“…Plasminogen activator (Plau) upregulated at 84 h-96 h-144 h is a growth factor, mitogen and apoptotic reducer [77,78]. Another upregulated gene (84 h-96 h) associated with cell growth, survival and transcription is the tumor-suppressor ras association domain-containing protein 6 (Rassf6) an important regulator of cell cycle arrest and apoptosis and whose upregulated family members were observed in high producers [55,79]. The transcription factor aryl hydrocarbon receptor nuclear translocator 2 (Arnt2) correlated with cell proliferation [80] was downregulated at 72 h-84 h. At 84 h-96 h Dnase1l3 and Egfr were upregulated encoding deoxyribonuclease gamma (Dnase1l3) and epidermal growth factor receptor (EGFR), respectively.…”

Chlorella vulgaris Extract as a Serum Replacement That Enhances Mammalian Cell Growth and Protein Expression

“…This is due to their innate membranous organelles allowing post-translational modification (PTM) of the expressed proteins, which is critical for certain proteins to fold properly and/or exert its biological activity (Amann et al, 2019). In particular, given their ease of in vitro cell culture and ability to adapt to selection pressure, Chinese hamster ovary (CHO) cells are established as the workhorse for industrial protein production (Berger et al, 2020).…”

Influence of cytoskeleton organization on recombinant protein expression by CHO cells

“…Further analysis of infliximab-producing ACTC1-overexpressing cell pools showed a significantly increased IgG titer after 10 days of fed-batch culture (Figure 2d). A positive effect of TAGAP on recombinant protein expression was also observed (see accompanying paper, Berger et al, 2020). | 1121 ACTC1 high expressor clones were randomly picked for further analysis ( Figure S3a).…”