Use of an anti‐apoptotic CHO cell line for transient gene expression

Abstract

Transient gene expression in mammalian cells allows for rapid production of recombinant proteins for research and preclinical studies. Here, we describe the development of a polyethylenimine (PEI) transient transfection system using an anti-apoptotic host cell line. The host cell line, referred to as the Double Knockout (DKO), was generated by deleting two pro-apoptotic factors, Bax and Bak, in a CHO-K1 cell line using zinc finger nuclease mediated gene disruption. Optimized DNA and PEI volumes for DKO transfections were 50% and 30% lower than CHO-K1, respectively. During transfection DKO cells produced relatively high levels of lactate, but this was mitigated by a temperature shift to 31°C which further enhanced productivity. DKO cells expressed ∼3- to 4-fold higher antibody titers than CHO-K1 cells. As evidence of their anti-apoptotic properties post-transfection, DKO cells maintained higher viability and had reduced levels of active caspase-3 compared to CHO-K1 cells. Nuclear plasmid DNA copy numbers and message levels were significantly elevated in DKO cells. Although DNA uptake levels, as early as 40 min post-transfection, were higher in DKO cells this was not due to differences in cell surface heparan sulfate (HS) or initial endocytosis mechanism as both cell types utilized caveolae- and clathrin-mediated endocytosis to internalize DNA:PEI complexes. These results suggest that the increased transfection efficiency and titers from DKO cells are attributed to their resistance to transfection-induced apoptosis and not differences in endocytosis mechanism.

A mechanistic dissection of polyethylenimine mediated transfection of CHO cells: To enhance the efficiency of recombinant DNA utilization

“…In this case, a submaximal polyplex‐mediated transfection and associated recombinant protein production per cell (Figure ) may be offset by superior long term cell viability (i.e., integral of viable cell concentration). However, use of a permissive CHO clone, intrinsically resistant to PEI cytotoxicity would permit higher cellular polyplex loads to be utilized to attain maximum production titer …”

“…Accordingly, reduction in ROS formation via transfection with a PEI‐derived amphiphilic copolymer containing the antioxidant α‐tocopherol was found to improve transfection efficiency in a range of cell lines . Engineered antiapoptotic cell lines have proved to be viable platforms for enhanced PEI mediated TGE . A future strategy to generate a cell line with superior resistance to the toxicity of PEI could follow a selective evolution method.…”

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

“…Different transfection reagents induce different levels of cytotoxicity in terms of impeded cell growth rate and a drop in viability (our own unpublished observations), as well as the inhibition of protein synthesis (Underhill et al, 2003). Notably, transfection stress can be reduced through process or cell engineering optimization (Johari et al, 2015;Macaraeg et al, 2013;Majors et al, 2008). In addition, variability in transfection efficiency is an inherent problem for TGE (Hansen et al, 2015;Liu et al, 2008); however, optimization and selection of the appropriate method can reduce variability substantially (Davies et al, 2013).…”

“…However, the lower protein yield achieved with TGE in CHO cells has historically been a major drawback compared to the substantially higher yields obtained with stable gene expression (SGE). Thus far, extensive efforts to improve TGE yields in CHO cells have been made by optimizing the culture environment (Galbraith et al, 2006;Ye et al, 2009), transfection efficiency (Mozley et al, 2014;Rajendra et al, 2015Rajendra et al, , 2012, vector systems (Cho et al, 2001;Mariati et al, 2010) and host cell line (Cain et al, 2013;Daramola et al, 2014;Macaraeg et al, 2013). Therefore, TGE has become a robust and flexible system, applicable to multiple r-proteins, expression volumes and bioprocesses with substantially increased yields of up to 3 g/L for MAbproducing CHO cells (Liu et al, 2015).…”

Transcriptional and post-transcriptional targeting for enhanced transient gene expression in CHO cells

“…The scalability and productivity of TGE in CHO cells have been significantly improved (Cain et al 2013;Steger et al 2014;Daramola et al 2014;Rajendra et al 2014). However, most approaches have resorted to cell line and vector engineering for enhancing transfectability of the CHO cells while also incorporating relatively long fed batch processes lasting up to 3 weeks (Cain et al 2013;Macaraeg et al 2013;Daramola et al 2014).…”