Effect of recombinant protein load on the activation of the unfolded protein response (UPR) in Chinese hamster ovary (CHO) cells


Background and novelty: The production of a recombinant protein (RP) in mammalian cells is regulated at multiple steps (1-4). Several laboratories have illustrated that RP production can be limited at one or more stages of the secretory pathway (5-7). This limitation has been characterised by the activation of the unfolded protein response (UPR) pathway and the presence of misfolded proteins in cells producing high amounts of RP (6, 7). The aim of this work is to profile of UPR component status for recombinant CHO cell lines which differ in terms of specific productivity. This data will be used to define settings of UPR gene expression that are associated with improved capacity to support complex protein production.

Experimental Approach: A series of clones of Chinese Hamster Ovary (CHO) cells, expressing erythropoietin (EPO), have been grown in serum-containing and CD protein-free medium as the experimental model. In some cases were subjected to methotrexate (250M) amplification. Clones were characterised in terms of growth, expression and structural variant profiles of intracellular and secreted EPO and UPR gene expression at mRNA level.

Results and Discussion: Recombinant CHO cell lines exhibited a 5-fold range of EPO specific productivities (Qp) between the amplified and non-amplified cell lines. Increased Qp was correlated with an increased intracellular content of EPO and the presence of high molecular weight intracellular EPO species were observed in the highest producing cell line. Despite the presence of abnormal intracellular EPO variants in cell lines with the highest Qp, there was no enhancement to the activation of the XBP-1 arm of the UPR and other genes profiled in this study showed similar dynamics for cell lines during batch culture. The accumulation of incompletely glycosylated EPO inside cells suggests that after passing a key quality assessment step fully glycosylated EPO is quickly secreted. This key post-translational modification presents a limiting step for effective EPO secretion and offers a potential target for cell host selection or genetic engineering.

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