Rational vector design and multi-pathway modulation of HEK 293E cells yield recombinant antibody titers exceeding 1 g/l by transient transfection under serum-free conditions

Abstract

Transient transfection allows for fast production of recombinant proteins. However, the current bottlenecks in transient transfection are low titers and low specific productivity compared to stable cell lines. Here, we report an improved transient transfection protocol that yields titers exceeding 1 g/l in HEK293E cells. This was achieved by combining a new highly efficient polyethyleneimine (PEI)-based transfection protocol, optimized gene expression vectors, use of cell cycle regulators p18 and p21, acidic Fibroblast Growth Factor, exposure of cells to valproic acid and consequently the maintenance of cells at high cell densities (4 million cells/ml). This protocol was reproducibly scaled-up to a working volume of 2 l, thus delivering >1 g of purified protein just 2 weeks after transfection. This is the fastest approach to gram quantities of protein ever reported from cultivated mammalian cells and could initiate, upon further scale-up, a paradigm shift in industrial production of such proteins for any application in biotechnology.

Figure 1.

Rational vector design tailored to HEK293E cells is essential for high-yield recombinant protein production. Comparison of different high-yield enhancer/promoters (human eIF1α, mouse CMV, human CMV) and regulatory elements (intron, WPRE). All elements were cloned into the same vector backbone of an IgG heavy chain and IgG light chain expression vector, respectively. We then transfected HEK293E cells with the corresponding constructs using the standard protocol and measured IgG yields via ELISA at day 5.

Figure 2.

Multi-pathway modulation of HEK293E cells in combination with step-wise optimization of a standard transfection protocol increases recombinant antibody titers by a factor of 27 from 40 to 1.1 g/l. (A) Comparison of the standard protocol to the XLG protocol. HEK293E cells were transfected as described in ‘Materials and Methods’ section. IgG titers were measured every day until cell viability dropped below 50%. (B and C) Results of ANOVA based on a design of experiments strategy in order to determine statistically significant independent and synergistic effects of the components (factors) of the XLG transfection protocol. The factors considered were cell density, p21, p18, aFGF and VPA. Full factorial experimental design (2⁵ = 32 experiments) was selected. Transfections were performed in triplicates as described in ‘Materials and Methods’ section. (B) Summary data of all 32 experiments. (C) Assumes VPA is always used, i.e. the VPA effect is included in the constant.

Figure 3.

The XLG protocol is scalable to the 2-l scale and specifically adapted to the pXLG^HEK vector backbone. (A) Results of large-scale transient transfection experiment with XLG protocol (2-l scale). Eight billion HEK293E cells were transfected in 400 ml FreeStyle medium according to the XLG Protocol (as described in ‘Materials and Methods’ section). Titers and cell viability were determined daily over a 10-day period. (B) Comparison of pEAK8 and pXLG^HEK vector backbone when used in combination with vectors coding for p18h, p21h and with and without a FGF.