High‐density transfection with HEK‐293 cells allows doubling of transient titers and removes need for a priori DNA complex formation with PEI

Abstract

Recombinant proteins are of great commercial and scientific interest. Yet, most production methods in mammalian cells involve the time- and labor-consuming step of creating stable cell lines. Production methods based on transient gene expression are advantageous in terms of speed and versatility; yet, depending on the transfection protocol, transient transfection faces some bottlenecks such as a priori complex formation, limitations in terms of transfection and production media used and the need for medium exchange prior to and/or after transfection. Published protocols for transfection of suspension-adapted HEK-293 cells with polyethyleneimine have shown great promise in overcoming some of these bottlenecks, but still require a priori complex formation for optimal yields and limit the choice of transfection and production media. Here, we report successful in situ transfection of suspension-adapted HEK-293 cells with 25-kDa linear polyethyleneimine at densities up to 20 x 10(6) cells/mL in complex media followed by production at lower cell densities (1 x 10(6) cells/mL). After concentrating cells to such high densities, transfection of HEK-293 cells becomes possible in most commonly used media and is not restricted to a specific medium. Furthermore, there is no need to make transfection complexes a priori, a step that prevents inline sterile filtration of the DNA bulk for transfection, an important consideration when scaling processes up to 100 or 1,000 L. Finally, transfecting HEK-293 cells at high density in complex media is superior to existing transfection protocols and doubles yields of recombinant protein obtainable by transient gene expression.

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

“…Compared with the standard two-step process, which requires an additional sterile vessel to premix PEI and DNA and then transfers the content into a bioreactor without sterile filtration (PEI/DNA particles cannot be sterile filtered), in situ complex formation is more amenable to upscaling and GMP compliance because of the lower contamination risk and easier handling involved. Research has suggested that in situ PEI/DNA complex formation can achieve higher reproducibility as small changes in maturation time can have a significant impact on titers (Backliwal et al 2008). In the present study, a higher transfection efficiency was obtained with the one-step transfection procedure.…”

“…A new and simpler method of TGE without priori formation of the PEI/DNA complex has been reported recently, and it has been confirmed to be better than a priori complex formation under certain conditions (Backliwal et al 2008). In the present study, we found that a one-step transfection process could produce higher transfection efficiency compared with the standard two-step process; however, there is no significant difference in GFP expression between these methods.…”

“…However, the environment of PEI/DNA formation is very different from the resting state, indicating that the parameters affecting transfection in the one-step procedure may be different from those of the standard protocol. In fact, research has shown that in situ complex formation outperforms a priori complex formation only at higher cell densities and higher concentrations of PEI and DNA (Backliwal et al 2008). In our study, cell viability in the one-step group was lower than that in the two-step group; however, these groups exhibited the same total GFP expression, although the former group produced a higher transfection efficiency (Fig.…”

“…Apart from the size of the complex, the overall charge of PEI/DNA particles is another parameter that affects transfection efficiency. Moreover, different PEI/DNA ratios could change the complex charge (Davis et al 1993;Kircheis et al 2001;Backliwal et al 2008). The interaction and stability of all PEI/DNA complexes were confirmed by gel retardation assay.…”

“…High PEI concentrations could not only produce compact particles with small sizes but also increase the quantity of PEI bound to DNA, thus increasing the particle's positive charge, which helps facilitate entry into cells and nuclei (Bertschinger et al 2006). Furthermore, the excess of bound PEI may enhance the efficiency of endosomal escape by PEI/DNA particles (Backliwal et al 2008). The results of the present study revealed that more DNA entered the cells and nuclei when the PEI ratio was higher and that many uncomplexed PEI molecules also permeated the nuclear membrane (Figs.…”