Enhanced transient recombinant protein production in CHO cells through the co‐transfection of the product gene withBcl‐x_L

Abstract

Transient gene expression is gaining popularity as a method to rapidly produce recombinant proteins in mammalian cells. Although significant improvements have been made, in terms of expression, more improvements are needed to compete with the yields achievable in stable gene expression. Much progress has come from optimization of transfection media and parameters, as well as altering culturing conditions to enhance productivity. Recent studies have included using cell lines engineered for apoptosis resistance through the constitutive expression of an anti-apoptotic protein, Bcl-xL. In this study we examine an alternative method of using the benefits of anti-apoptotic gene expression to enhance the transient expression of biotherapeutics, namely, through the co-transfection of bcl-xL and the product-coding gene. CHO-S cells were co-transfected with the product-coding gene and a vector containing Bcl-xL using polyethylenimine. Cells co-transfected with Bcl-xL showed reduced levels of apoptosis, increased specific productivity, and an overall increase in product yield of approximately 100%. Similar results were produced by employing another anti-apoptotic protein, Bcl-2 delta in CHO cells, or through the co-transfection with bcl-xL using HEK-293E cells. This work provides an alternative method for increasing yields of therapeutic proteins in TGE applications without generating a prior stable cell line and subsequent screening which are both time and resource consuming.

Disruption of the gene C12orf35 leads to increased productivities in recombinant CHO cell lines

“…With the recently published CHO genomes (Brinkrolf et al, 2013;Lewis et al, 2013;Xu et al, 2011) and advances in cell line engineering tools (Cheng and Alper, 2014) many studies focus on various high throughput "omics technologies" with the aim to identify molecular targets for cell line engineering approaches as well as for gaining a better understanding of cellular processes leading to higher productivities (Datta et al, 2013;Farrell et al, 2014;Gutierrez and Lewis, 2015;Kildegaard et al, 2013). Transcription, translation, protein folding, protein modification and protein export as well as regulation of cell cycle, apoptosis, cell growth, metabolic pathways, or epigenetic modifications could act as possible intervention points (Delic et al, 2014;Dickson, 2014;Du et al, 2015;Farrell et al, 2014;Jadhav et al, 2013;Xiao et al, 2014;Yang et al, 2014;Zustiak et al, 2014).…”

OPP Labeling Enables Total Protein Synthesis Quantification in CHO Production Cell Lines at the Single‐Cell Level

“…More detailed understanding of these mechanisms and correlations may enable the design of engineering strategies that target the translational machinery directly. Protein or miRNA based strategies, such as the activation of the mTOR signaling pathway or overexpression of translation initiation factors, might help to overcome the “bottleneck of translation” some host cell lines might face, and thus lead to the development of new hosts with improved capacity for growth and productivity . Recently, Ge et al reported the synthesis of puromycin analogues suitable for click chemistry in live cells, which would allow adaptation of this method for cell sorting.…”

Oxidative stress‐alleviating strategies to improve recombinant protein production in CHO cells

“…Considerable effort has been put into developing strategies to reduce apoptosis, with several efforts resulting in increased viability and, indirectly, improved process productivity (Meents, Enenkel, Eppenberger, Werner, & Fussenegger, 2002). Notably, the overexpression of bcl-2 or bcl-x L has been successfully used to activate antiapoptotic pathways in CHO cells (Tey, Singh, Piredda, Piacentini, & Al-Rubeai, 2000;Zustiak, Jose, Xie, Zhu, & Betenbaugh, 2014). A similar approach can be considered to relieve oxidative stress by increasing cellular defense or decreasing ROS-generating cellular activities.…”