Hyperosmotic stimulus study discloses benefits in ATP supply and reveals miRNA/mRNA targets to improve recombinant protein production of CHO cells

Abstract

Biopharmaceuticals are predominantly produced by Chinese hamster ovary (CHO) cells cultivated in fed-batch mode. Hyperosmotic culture conditions (≥ 350 mOsmol kg(∑1) ) resulting from feeding of nutrients may enhance specific product formation rates (qp ). As an improved ATP supply was anticipated to enhance qp this study focused on the identification of suitable miRNA/mRNA targets to increase ATP levels. Therefor next generation sequencing and a compartment specific metabolomics approach were applied to analyze the response of an antibody (mAB) producing CHO cell line upon osmotic shift (280 → 430 mOsmol kg(-1) ). Hyperosmotic culture conditions caused a ∼2.6-fold increase of specific ATP formation rates together with a ∼1.7-fold rise in cytosolic and mitochondrial ATP-pools, thus showing increased ATP supply. mRNA expression analysis identified several genes encoding glycosylated proteins with strictly tissue related function. In addition, hyperosmotic culture conditions induced an upregulation of miR-132-3p, miR-132-5p, miR-182, miR-183, miR-194, miR-215-3p, miR-215-5p which have all been related to cell cycle arrest/proliferation in cancer studies. In relation to a previous independent CHO study miR-183 may be the most promising target to enhance qp by stable overexpression. Furthermore, deletion of genes with presumably dispensable function in suspension growing CHO cells may enhance mAB formation by increased ATP levels.

Keywords: Adenine nucleotide pools; Cell line engineering targets; Chinese hamster ovary (CHO) cells; Hyperosmotic stress; Recombinant protein production.