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

Abstract

Rosmarinic acid (RA) has gained attraction in bioprocessing as a media supplement to improve cellular proliferation and protein production. Here, we observe up to a two‐fold increase in antibody production with RA‐supplementation, and a concentration‐dependent effect of RA on cell proliferation for fed‐batch CHO cell cultures. Contrary to previously reported antioxidant activity, RA increased the reactive oxygen species (ROS) levels, stimulated endoplasmic reticulum (ER) stress, activated the unfolded protein response (UPR), and elicited DNA damage. Despite such stressful events, RA appeared to maintained cell health via mTOR pathway activation; both mTORC1 and mTORC2 were stimulated in RA‐supplemented cultures. By reversing such mTOR pathway activity through either chemical inhibitor addition or siRNA knockdown of genes regulating the mTORC1 and mTORC2 complexes, antibody production, UPR signaling, and stress‐induced DNA damage were reduced. Further, the proliferative effect of RA appeared to be regulated selectively by mTORC2 activation and have reproduced this observation by using the mTORC2 stimulator SC‐79. Analogously, knockdown of mTORC2 strongly reduced XBP1 splicing, which would be expected to reduce antibody folding and secretion, sugging that reduced mTORC2 would correlate with reduced antibody levels. The crosstalk between mTOR activation and UPR upregulation may thus be related directly to the enhanced productivity. Our results show the importance of the mTOR and UPR pathways in increasing antibody productivity, and suggest that RA supplementation may obviate the need for labor‐intensive genetic engineering by directly activating pathways favorable to cell culture performance.This article is protected by copyright. All rights reserved

Advances in serum‐free media for CHO cells: From traditional serum substitutes to microbial‐derived substances

“…Therefore, the elimination of these by‐products and the supplementation of bioactive compounds such as rosmarinic acid during fed‐batch cultures have been shown to significantly enhance CHO cell proliferation in a concentration‐dependent manner. [ 23,24 ] In addition, increasing iron concentration in the culture medium enhances protein titers during biopharmaceutical process development; however, overdose of iron in the medium often leads to unacceptable protein discoloration. Xu et al.…”