Transient Transfection Factors for High-Level Recombinant Protein Production in Suspension Cultured Mammalian Cells

Abstract

The efficient transfection of cloned genes into mammalian cells system plays a critical role in the production of large quantities of recombinant proteins (r-proteins). In order to establish a simple and scaleable transient protein production system, we have used a cationic lipid-based transfection reagent-FreeStyle MAX to study transient transfection in serum-free suspension human embryonic kidney (HEK) 293 and Chinese hamster ovary (CHO) cells. We used quantification of green fluorescent protein (GFP) to monitor transfection efficiency and expression of a cloned human IgG antibody to monitor r-protein production. Parameters including transfection reagent concentration, DNA concentration, the time of complex formation, and the cell density at the time of transfection were analyzed and optimized. About 70% GFP-positive cells and 50-80 mg/l of secreted IgG antibody were obtained in both HEK-293 and CHO cells under optimal conditions. Scale-up of the transfection system to 1 l resulted in similar transfection efficiency and protein production. In addition, we evaluated production of therapeutic proteins such as human erythropoietin and human blood coagulation factor IX in both HEK-293 and CHO cells. Our results showed that the higher quantity of protein production was obtained by using optimal transient transfection conditions in serum-free adapted suspension mammalian cells.

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

“…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). For the SEE, IE and SGE platforms, transfection stress is most likely not an issue because of the lengthy (≥2 weeks) antibiotic-based selection processes.…”

Stable and Transient Expression of Human Coagulation Factor IX in Mammalian Expression Systems; CHO Versus HEK Cells

“…Liu et al showed that the EPO specific mRNA expressed in transient HEK cells remained constant, whereas the EPO mRNA level has been dramatically reduced in CHO cells after 4 days post-transfection (6). On the other hand, although a threefold elevation of EPO mRNA level was expressed in HEK cells compared to CHO cells, the maximal accumulations of EPO were greater in FreeStyle TM CHO, being sevenfold higher than the maximum accumulations obtained from FreeStyle TM HEK cells (6). It could be due to the lower secretion efficiency of HEK cells rather than that of CHO cells.…”

“…HEK cells have generated the highest volumetric yields in TGE systems, and are therefore the most widely used (23). For many applications, the use of transient HEK, rather than CHO cells, appears to be superior, since HEK cells are 6 Gene Cell Tissue. 2017; 4(2):e13096.…”

Effect of addition of ‘carrier’ DNA during transient protein expression in suspension CHO culture

“…Prior to evaluating the effect of carrier DNA, we tested the effect of these factors on culture productivity. DNA delivery agents such as calcium phosphate, liposomes and cationic polymers like polyethyleneimine (PEI) have been used to transfect 1 ml to 100 l suspension cultures of mammalian cells to transiently express recombinant proteins (Derouazi et al 2004;Girard et al 2002;Liu et al 2008;Schlaeger and Christensen 1999;Meissner et al 2001;Muller et al 2007;Schlaeger et al 2003). Polyethyleneimine is one of the widely used transfection agents used due to its ability to transfect a broad range of cell lines and low toxicity along with its cost effectiveness over commercially available reagents which could be an important consideration for scale up (Boussif et al 1995).…”