CHOgro® Expression Medium Yields Multi-fold Increases in Antibody Titer. Human IgG1 was produced by transient transfection using TransIT-PRO® (1:1), FreeStyle™ MAX (1.25:1.25)  or 25kDa linear PEI (6:1) transfection reagents according to the manufacturers’ or published protocol (reagent:DNA ratio). Transfections were performed using 1 µg plasmid DNA per milliliter of culture and cell densities of 2 x 10⁶ cells/ml or 1x 10⁶ cells/ml  for the CHOgro® Expression Medium (red bars) or FreeStyle™  Expression Medium (blue bars), respectively, at the time of transfection.  FreeStyle™ CHO-S cells were cultured in CHOgro® Expression Medium or FreeStyle™ CHO Expression Medium  and plated into non-treated 6-well plates (2ml/well) for transfection. Antibody levels were also analyzed from day 6 clarified supernatants using a human IgG ELISA (ZeptoMetrix). Error bars represent the standard deviation of triplicate technical replicates.

High Efficiency Transfection of CHO-S Cells Using the TransIT-PRO® Transfection Reagent. Plasmid DNA encoding EGFP was delivered by transient transfection with the TransIT-PRO® Transfection Reagent (1:1) (reagent:DNA ratio, volume:weight) using 1 µg plasmid DNA per milliliter of culture and cell a density of 2 x 10⁶ cells/ml in the CHOgro® Expression Medium at the time of transfection.  FreeStyle™ CHO-S cells were cultured in CHOgro® Expression Medium  and plated into non-treated 6-well plates (2 ml/well) for transfection. (A) GFP levels and cell viability (propidium iodide) were measured 48 hours post-transfection using a Guava® easyCyte™ 5HT flow cytometer (EMD Millipore). (B) Images were captured using a Zeiss Axiovert inverted fluorescence microscope.

Titers of Five Different Antibody Vector Constructs Using the CHOgro® Expression System. Five different antibody constructs were produced by transient transfection using TransIT-PRO® at a 1:1 reagent:DNA ratio. Transfections were performed using 1 µg plasmid DNA per milliliter of culture and a cell density of 2 x 10⁶ cells/ml at the time of transfection.  FreeStyle™ CHO-S cells were cultured in CHOgro® Expression Medium and seeded into 30 ml media in 125 ml shake flasks (Thomson) for transfection. Day 11 supernatants were clarified and analyzed using a human IgG ELISA (ZeptoMetrix). Error bars represent the standard deviation of triplicate technical replicates.

Increases in Product Titer are Observed at Longer Time Points with Mild Hypothermic Conditions. Human IgG1 was produced by transient transfection with the TransIT-PRO® Transfection Reagent and 1 µg plasmid DNA per milliliter of culture at a 1:1 reagent:DNA ratio. Cells were transfected at a density of 2 x 10⁶ cells/ml in 20 ml of CHOgro® Expression Medium in 125 ml shake flasks (Thomson). Antibody levels were also analyzed from day 4, 7 and 11 clarified supernatants using a human IgG ELISA (ZeptoMetrix). All flasks were incubated at 37°C for 24 hours; at that point designated parallel flasks were switched to 32°C for the remainder of the experiment. Error bars represent the standard deviation of triplicate technical replicates.