Insights into the Impact of Rosmarinic Acid on CHO Cell Culture Improvement through Transcriptomics Analysis

Abstract

The use of antioxidants in Chinese hamster ovary (CHO) cell cultures to improve monoclonal antibody production has been a topic of great interest. Nevertheless, the antioxidants do not have consistent benefits of production improvement, which might be cell line specific and/or process specific. In this work, we investigated how treatment with the antioxidant rosmarinic acid (RA) improved cell growth and titer in CHO cell cultures using transcriptomics. In particular, transcriptomics analysis indicated that RA treatment modified gene expression and strongly affected the MAPK and PI3K/Akt signaling pathways, which regulate cell survival and cell death. Moreover, it was observed that these signaling pathways, which had been identified to be up-regulated on day 2 and day 6 by RA, were also up-regulated over time (from initial growth phase day 2 to slow growth or protein production phase day 6) in both conditions. In summary, this transcriptomics analysis provides insights into the role of the antioxidant RA in industrial cell culture processes. The current study also represents an example in the industry of how omics can be applied to gain an in-depth understanding of CHO cell biology and to identify critical pathways that can contribute to cell culture process improvement and cell line engineering.

Rosmarinic acid enhances CHO cell productivity and proliferation through activation of the unfolded protein response and the mTOR pathway

“…To confirm the use of RA to prolong cell culture duration, we supplemented fed-batch grown CHOZN antibody-expressing cells and compared to unsupplemented, control cultures. Similar to previous reports, [20,37] we observed a proliferative effect of RA on fed-batch cultures. Supplementation of RA elongated the viable cell period (defined as total days in a fed-batch culture when cell viability is above 70%) from 8 days in untreated culture to 10 days with 0.1 mM RA supplementation and 14 days with 0.5 mM supplementation (Figure 1A).…”

“…The balance between the three governs the oxidative environment in media and would require special attention when making commercial media. However, the proliferative effects of RA is consistent to that observed in other cell lines under a variety of conditions, [ 37 ] suggesting RA's primary mechanism of action is not through oxidative stress alleviation, but alternative pathway stimulation.…”

“…[20,44] Huang et al showed RA addition did not result in changes in SOD1 levels, suggesting RA did not activate cellular pathways associated with ROS removal. [37] We observed a similar effect on antibody production and cell growth (Figure 1) but detected an increased level of ROS in both the cell culture media and intracellularly (Figures 2 and 4). RA, along with other phenolic compounds, has been conventionally viewed as an oxidative stress alleviator that acts to stabilize the reactive oxygen radical via electron acceptance or hydrogen transfer.…”

“…First, we determined cell cycle differences using propidium iodide staining and flow cytometry analysis and observed S and G2/M cell cycle arrest (Figure 4A, Figure S3) in RA‐supplemented cells as indirect evidence of DNA damage. [ 37 ] To further test G2/M cell cycle arrest and the related DNA damage effect, we quantified the levels of GADD45a mRNA. GADD45a is a pro‐survival gene that inhibits CDK1/CycB activation directly to lead to G2/M cell cycle arrest and reduced cell growth.…”

“…RA has been shown to upregulate gene transcription in the MAPK pathway [ 37 ] that leads to ROS‐signaling, cell cycle progression and pro‐survival effects. [ 83 ] However, we show selective upregulation of the Akt/mTOR pathway (Figure 5), but not the MAPK pathway (Figure S8).…”