Effect of inducible co‐overexpression of protein disulfide isomerase and endoplasmic reticulum oxidoreductase on the specific antibody productivity of recombinant Chinese hamster ovary cells

Abstract

To enhance specific antibody (Ab) productivity (q(Ab)) of recombinant Chinese hamster ovary (rCHO) cells, post-translational limitations in the endoplasmic reticulum during antibody production should be relieved. Previously, we reported that overexpression of protein disulfide isomerase (PDI), which catalyzes disulfide bond exchanges and assists in protein folding of newly synthesized proteins, enhanced q(Ab) of rCHO cells by about 27% (Mohan et al., 2007, Biotechnol Bioeng 98:611-615) . Since the rate limiting step in disulfide bond formation is found to be the regeneration of oxidized PDI, the oxidation state of PDI, as well as the amount of PDI, might be important. Endoplasmic reticulum oxidoreductase (ERO1L) maintains PDI in an oxidized state so that disulfide bond formation occurs. Here, PDI and its helper protein, ERO1L were overexpressed in rCHO cells producing an Ab in an attempt to ease the bottleneck in disulfide bond formation, and hence, Ab folding and secretion. Transient expression of ERO1L alone and with PDI resulted in enhanced q(Ab) by 37% and 55%, respectively. In contrast, under stable inducible co-overexpression of PDI and ERO1L, the q(Ab) was unaffected or negatively affected by varying degrees, depending on the individual expression levels of these genes. In stable clones with altered oxidation state of PDI due to co-overexpression of PDI and ERO1L, secretion of Ab was hindered and PDI-associated retention of Ab was seen in the cells. Under transient gene expression, secretion of Ab was not compromised. The data presented here suggests a possible mechanism of PDI/ERO1L interaction with the target Ab and shows how the expression levels of these proteins could affect the q(Ab) of this Ab-producing rCHO cell line.

Improving the secretory capacity of Chinese hamster ovary cells by ectopic expression of effector genes: Lessons learned and future directions

“…For example, effects obtained from TGE are sometimes transferable to a SGE context (Tastanova et al, 2015) and sometimes not (Mohan and Lee, 2010). The need for validation is obviously a drawback of the TGE, SEE and IE platforms.…”

“…1). Several reports have demonstrated synergistic effects on the volumetric productivity or q p of co-expressing multiple genes in CHO cells (Cain et al, 2013;Le Fourn et al, 2014;Mohan and Lee, 2010;Peng and Fussenegger, 2009), demonstrating the potential of combining effector genes. In addition to effector gene overexpression, downregulating endogenous genes might be equally important for increasing q p (Feichtinger et al, 2016;Harreither et al, 2015).…”

“…c) Inducible expression of a stably integrated gene encoding the r-protein in a clonally derived cell line constitutively expressing an effector gene of interest. No reported examples of this specific setup are available to the best of the authors' knowledge, but examples of the expression system are described here: (Mohan et al, 2007;Mohan and Lee, 2010). d) Constitutive (stable) co-expression of stably integrated genes encoding r-protein and effector genes in clonally derived cell lines.…”

3.5 Manipulation of Cell Growth, Metabolism and Product Quality Attributes

“…Overexpression of protein disulfide-isomerase even decreased target protein secretion [87]. Later it was found that the oxidative state of PDI is important for activity and an expression system for inducible coexpression of PDI was devised [88].…”

Experimental and In Silico Modelling Analyses of the Gene Expression Pathway for Recombinant Antibody and By-Product Production in NS0 Cell Lines

“…Particular targets investigated to date with a view to improving qP in mammalian cell lines include anti-apoptotic genes [10], [11], [12], [13], cell cycle related genes [14], [15], [16], the folding and assembly machinery in the endoplasmic reticulum [17], [18], [19], [20], [21], [22], and the translational [23], [24], [25] and secretory machinery [26]. However, such approaches to improving qP in mammalian cell lines have largely resulted in conflicting or disappointing results.…”