Effect of Peptone Feeding on Transient Gene Expression Process in CHO DG44

Abstract

Background: Transient Gene Expression (TGE) gained popularity over the last decade as a rapid method for the production of milligram to gram quantities of recombinant proteins for preclinical studies in biophama industry. Thereby, the optimization of the TGE technique for Chinese hamster ovary (CHO) as the dominant host for the production of biotherapeutics is of great interest to reach the values for Human Embryo Kidney-293 (HEK-293) cells in terms of transfection efficiencies and production titers. TGE efficiencies are cell line and vector dependant.

Methods: In transfection efficiency optimization experiments, different starting cell densities, different amounts of plasmid DNA and PEI transfection reagent were investigated to achieve the best conditions leading to maximum transfection efficiencies. Furthermore, in order to investigate the effect of peptone feeding on transfection efficiency, three different sources of peptones with the greatest effect in the CD DG44 basal media were selected; Casein Tryptone N1, Soy petone A2SC and Soy peptone E110.

Results: The transfection strategy performed here was able to make an outstanding increase in transfection efficiency of CHO DG44 cell line transfected with pTracer-SV40-mutated t-PA plasmid from 3.6% in our starting non-optimized condition to 66.93% in finally optimized situation. Moreover, peptone feeding strategy used here was successful to increase volumetric productivities up to 37%. In addition, the amounts of both PEI and plasmid DNA were reduced up to 66% and 25%, respectively compared to our previous protocol.

Conclusion: Here we described an optimization process for TGE in suspension-adapted CHO cells based on Polyethylenimine (PEI)/DNA concentration, DNA: PEI ratio, starting cell densities and peptone feeding strategy.

Keywords: Chinese hamster ovary cells; Peptones; Polyethylenimine; Protein production; Transient gene expression.

Figure 1

Optimization of PEI amount. Varying amounts of PEI were added as indicated with a constant DNA amount of 1.25 µg/10⁶ cells and a starting cell density of 2×10⁶cells/ml. A) GFP positive cells 48 hr posttransfection with different PEI concentrations; B) t-PA recombinant protein concentration in the culture media measured on day 9 posttransfection by ELISA

Figure 2

Optimization of the starting cell density for transfection. Varying starting cell densities from 0.2 to 4×10⁶cells/ml with constant DNA and PEI concentrations were investigated. A) GFP positive cells 48 hr posttransfection with different starting cell densities; B) t-PA recombinant protein concentration in the culture media measured on day 9 posttransfection by ELISA

Figure 3

Optimization of DNA concentration for transfection. Varying DNA concentrations from 0.25 to 1.25 µg/10⁶ cells with 0.5×10⁶ starting cell densities and 1.5 µg/10⁶ cells of PEI concentrations were investigated. A) GFP positive cells 48 hr posttransfection with different DNA plasmid concentrations; B) t-PA recombinant protein concentration in the culture media measured on day 9 posttransfection by ELISA

Figure 4

Effect of three different peptones on transfection efficiency and production yield. 1 g/l of final concentrations of peptones were added to the transfection reaction with 0.5 µg/10⁶ cells of DNA, 0.5×10⁶ starting cell density and 1.5 µg/ 10⁶ cells of PEI concentrations. A) GFP positive cells 48 hr posttransfection with different peptone feeding strategies; B) t-PA recombinant protein concentration in the culture media measured on day 9 posttransfection by ELISA

Figure 5

Effect of stepwise optimization on transfection efficiencies of CHO DG44 cells transiently transfected with pTracer-SV40-mutated t-PA plasmid. The optimized values for each step were used in the next experiment