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

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. A key premise for secretion of a recombinant protein is to get the recombinant mRNA to efficiently assimilate into the endogenous mRNA pool of ER‐directed mRNAs. By applying subcellular fractionation, the subcellular distribution between the cytosol and the ER surface of recombinant mRNAs encoding EPO and Rituximab during transient expression in CHO cells is examined. It is shown that the recombinant mRNAs display subcellular localization equal to that of endogenous ER‐directed mRNA. Furthermore, it is shown that these results can be transferred to a stable CHO cell line expressing high levels of recombinant mRNA. These results indicate that, under conditions described, the cells capability to direct most recombinant mRNA to the surface of the ER is not fully saturated.