Balancing GFP reporter plasmid quantity in large-scale transient transfections for recombinant anti-human Rhesus-D...

Using transient expression, high amounts (>20 mg/mL) of secreted anti-human Rhesus-D IgG1 were produced in a suspension-adapted HEK293 EBNA cell line (Meissner et al., Biotechnol Bioeng 75: 197-203, 2001). Time of harvest was 3 days after transfection. For the estimation of transfection efficiencies, we routinely co-transfected EGFP reporter DNA. At higher reporter plasmid concentrations, >2% of total transfecting plasmid DNA, a substantial reduction of recombinant antibody synthesis, was observed. This phenomenon was investigated in detail by co-expressing various green fluorescent protein (GFP) reporter constructs, which were targeted at different subcellular locations. Enhanced and humanized GFPs targeted to either the endoplasmic reticulum, the cytosol, or the nucleus reduced recombinant antibody production by 30 to 40% when present at higher concentrations in the transfection solution. The most severe effects were observed when the co-transfected EGFP was targeted to the endoplasmic reticulum, leading to a reduction of up to 80% in the presence of only 5% of reporter DNA. Interestingly, one nuclear-targeted GFP variant that was not codon optimized for expression in human cell lines could be added, to up to almost half of the total amount of transfecting DNA, without adverse effect on antibody production. Although the minimum amount of this reporter DNA needed for fluorescence reading was 10 times higher than for the other variants, it provided a much broader quantity range within which the transfection process could be studied without being negatively affected.