Role of non‐specific DNA in reducing coding DNA requirement for transient gene expression with CHO and HEK‐293E cells

Abstract

Transient gene expression (TGE) is a rapid method for the production of recombinant proteins in mammalian cells. While the TGE volumetric productivity has improved significantly over the past decade, the amount of plasmid DNA (pDNA) needed for transfection remains very high. Here, we examined the use of non-specific (filler) DNA to partially replace the transgene-bearing plasmid DNA (coding pDNA) in transfections of Chinese hamster ovary (CHO) and human embryo kidney (HEK-293E) cells. When the optimal amount of coding pDNA for either host was reduced by 67% and replaced with filler DNA, the recombinant protein yield decreased by only 25% relative to the yield in control transfections. Filler DNA did not affect the cellular uptake or intracellular stability of coding pDNA, but its presence lead to increases of the percentage of transfected cells and the steady-state level of transgene mRNA compared to control transfections. Studies of the physicochemical properties of DNA-polyethyleneimine (PEI) complexes with or without filler DNA did not reveal any differences in their size or surface charge. The results suggest that filler DNA allows the coding pDNA to be distributed over a greater number of DNA-PEI complexes, leading to a higher percentage of transfected cells. The co-assembly of filler DNA and coding pDNA within complexes may also allow the latter to be more efficiently utilized by the cell's transcription machinery, resulting in a higher level of transgene mRNA.

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

“…Filler plasmids are used to titrate the effector gene-encoding plasmid Rajendra et al, 2015Rajendra et al, , 2012, whereas promotors with different strengths drive transcription at different rates (Brown and James, 2015;Qin et al, 2010). The filler plasmid principle is a cost-efficient solution in transient expression-based setups, as no additional cloning work is needed besides a single plasmid preparation of the filler plasmid.…”

“…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).…”

“…In terms of gene dosage titration (Rajendra et al, 2012) and combining genes , TGE offers complete flexibility, as a variety of different plasmids (filler plasmid and/or plasmids carrying different effector genes) can be coexpressed. This is not the case for SEE, as the copy numbers of two plasmids with different genes expressed, in some cases, will drift towards selection for the plasmid/gene giving rise to the lowest fitness cost (personal communication with Dr Mikael Rørdam Andersen, Technical University of Denmark).…”

Direct Interrogation of DNA Content Distribution in Nanoparticles by a Novel Microfluidics-Based Single-Particle Analysis

“…Other groups have also reported similar observations. 9,10 These studies highlight the limitations of our current state of understanding of a complex process and the need for a more quantitative and detailed analysis of nanoparticle compositions. By enabling rapid DNA content distribution, our method will be an invaluable tool to accomplish this.…”

Cell Culture Processes for Biopharmaceutical Manufacturing

“…We have showed that the supplementation of plasmid DNA with non-coding DNA to increase extracellular DNA concentration, increases the transfection efficiency and TGE yield per unit plasmid 33 . Such an increase was shown to be accompanied by an increase in transgene mRNA levels 34 . After the initial delivery of plasmid to the cell, plasmid copy numbers continuously decrease due to dilution by cell division resulting in decrease in protein expression.…”