Endoplasmic reticulum‐directed recombinant mRNA displays subcellular localization equal to endogenous mRNA during transient expression in CHO cells

Abstract

When expressing pharmaceutical recombinant proteins in mammalian cells, the protein is commonly directed through the secretory pathway, in a signal peptide-dependent manner, to acquire specific post-translational modifications and to facilitate secretion into the culture medium. One key premise for this is the direction of the mRNA encoding the recombinant protein to the surface of the endoplasmic reticulum (ER) for subsequent protein translocation into the secretory pathway. To evaluate the efficiency of this process in Chinese hamster ovary (CHO) cells, the subcellular localization of recombinant mRNA encoding the therapeutic proteins, erythropoietin (EPO) and Rituximab, was determined. The results show that ER-directed recombinant mRNAs exhibited an efficient recruitment to the ER when compared to an endogenous ER-directed mRNA, with no cytoplasmic translation of ER-directed recombinant proteins observed. These observations indicate that the recombinant mRNA, encoding ER-directed proteins, follows the same distribution pattern as endogenous mRNA directed towards the ER. Furthermore, the previous established fractionation method proves to be an efficient tool to study not only recombinant mRNA localization, but also recombinant protein trafficking between the ER and cytosol in CHO cells.

Ribosome profiling-guided depletion of an mRNA increases cell growth rate and protein secretion

“…This observation was supported by the findings that the specific productivity and transcript levels of the antibody showed a proportional relationship. Since degradation of the recombinant proteins and residence time in the secretory pathway are important factors in relation to the specific productivity38, future work could aim to explore how these factors work together to affect recombinant expression.…”

Secretion analysis of intracellular “difficult-to-express” immunoglobulin G (IgG) in Chinese hamster ovary (CHO) cells

“…Cell line engineering and cell culture engineering strategies have contributed to achieving very high protein titers (e.g., * 10 g/L) for some recombinant proteins (Wurm 2004;Li et al 2010;Bandaranayake and Almo 2014;Kunert and Reinhart 2016;Takagi et al 2017;Kuwae et al 2018). Various approaches to host cell engineering have also been studied at the genome (Omasa 2002;Kameyama et al 2010;Kawabe et al 2012Kawabe et al , 2017Wang et al 2017), DNA transcription (Lai et al 2013;Kawabe et al 2017), RNA translation (Le Fourn et al 2014;Haryadi et al 2015;Chng et al 2015;Kallehauge et al 2016), and protein modification and secretion levels (Borth et al 2005;Tigges and Fussenegger 2006;Peng and Fussenegger 2009;Pybus et al 2014a). Such cell modification strategies have increased the productivity of recombinant protein production.…”

Recombinant Human CD19 in CHO-K1 Cells: Glycosylation Patterns as a Quality Attribute of High Yield Processes

“…Difficult-to-express (DTE) proteins are often underrepresented in scientific and technological publications, since monoclonal antibodies (mAb) have become the dominant class in the biopharmaceutical market [ 1 , 6 , 7 , 8 , 9 ], possessing high expression levels [ 10 ], relatively simple glycosylation with only one N-glycosylation site that is practically non-sialylated [ 11 ], and established analysis and purification methods [ 12 ].…”