Serum‐free large‐scale transient transfection of CHO cells

Abstract

To date, methods for large-scale transient gene expression (TGE) in cultivated mammalian cells have focused on two transfection vehicles: polyethylenimine (PEI) and calcium phosphate (CaPi). Both have been shown to result in high transfection efficiencies at scales beyond 10 L. Unfortunately, both approaches yield higher levels of recombinant protein (r-protein) in the presence of serum than in its absence. Since serum is a major cost factor and an obstacle to protein purification, our goal was to develop a large-scale TGE process for Chinese hamster ovary (CHO) cells in the absence of serum. CHO-DG44 cells were cultivated and transfected in a chemically defined medium using linear 25 kDa PEI as a transfection vehicle. Parameters that were optimized included the DNA amount, the DNA-to-PEI ratio, the timing and solution conditions for complex formation, the transfection medium, and the cell density at the time of transfection. The highest levels of r-protein expression were observed when cultures at a density of 2.0 x 10(6) cells/ml were transfected with 2.5 microg/ml DNA in RPMI 1640 medium containing 25 mM HEPES at pH 7.1. The transfection complex was formed at a DNA:PEI ratio of 1:2 (w/w) in 150 mM NaCl with a 10-min incubation at room temperature prior to addition to the culture. The procedure was scaled up for a 20-L bioreactor, yielding expression levels of 10

Polyethylenimine-based nanocarriers in co-delivery of drug and gene: a developing horizon

“…The transfection process can be affected by many unknown factors. However, several studies have linked the transfection efficiency and cytotoxicity of PEI preparations to the physicochemical properties, the molecular weight and the branching ratio of polymer [41,42], the amount of DNA, the DNA ratio to the PEI, the timing and the solution conditions for complex formation, the transfection medium and cell density at time of transfer [43,44]. In addition, many factors, including temperature, surfactant, complex concentration, ionic strength, viscosity, pH, can significantly affect the aggregation process.…”

PEI/DNA formation affects transient gene expression in suspension Chinese hamster ovary cells via a one-step transfection process

“…Chinese hamster ovary (CHO) cells are widely used in the biotechnology industry to produce recombinant proteins (Derouazi et al 2004;Wurm and Bernard 1999). The most rapid and least expensive method for recombinant protein expression in mammalian cells is transient gene expression (TGE).…”

“…The most rapid and least expensive method for recombinant protein expression in mammalian cells is transient gene expression (TGE). This approach has made it possible to produce milligram-to-gram quantities of a recombinant protein within days or weeks (Wurm and Bernard 1999;Derouazi et al 2004). Its ability to generate proteins within a relatively short timeframe enables TGE to evaluate functionalities and toxicities much earlier and therefore provide valuable information about drug candidates (Girard et al 2002;Jordan et al 1996).…”

Iron (III) citrate inhibits polyethylenimine-mediated transient transfection of Chinese hamster ovary cells in serum-free medium

“…PEI condenses DNA into particulate complexes, which are then believed to be taken up by the cell through endocytosis (Kopatz et al 2004). Currently, most reports of transient transfection using PEI have used either HEK 293 or Chinese hamster ovary (CHO) cells (Derouazi et al 2004;Schlaeger and Christensen 1999;Tait et al 2004). It appears that higher titers may be achieved using 293 cells, due to the ability of these cells to utilize the EBNA protein for replicating plasmids (Shen et al 1995).…”