CHOgro^® Expression System

Fig. 1 | 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.

Fig. 2 | CHOgro^® Expression System Enables Broad Scalability, 1000-fold. 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 CHOgro^® Expression Medium on an orbital shaker at the following volumes/culture vessels: 2.5 ml/non-tissue culture treated 6-well dish, 25 ml/125 ml Thomson flask, 100 ml/250 ml Thomson flask, 500 ml/1.6 L Thomson flask, 2500 ml/5 L Thomson flask. At twenty-four hours post-transfection all cultures were moved to 32°C for the remainder of the experiment. Antibody levels were analyzed from day 7 clarified supernatants using a human IgG ELISA (Zeptometrix). All values are normalized to the 25 ml volume sample and error bars represent the standard error of the mean of triplicate technical replicates.

Fig. 3 | Higher Cell Densities Leads to Higher Titers Using the CHOgro^® Expression System. Human IgG1 was produced by transient transfection using TransIT-PRO^® (1:1) or FreeStyle™ MAX (1.25:1.25) transfection reagents according to the manufacturers' protocol (reagent:DNA ratio, volume:weight). Transfections were performed using 1 µg plasmid DNA per milliliter of culture and cell densities of 1, 2 or 4 x 10⁶ cells/ml at the time of transfection. FreeStyle™ CHO-S cells were cultured in CHOgro^® Expression Medium (red bars) or FreeStyle™ CHO Expression Medium (blue bars) and plated into non-treated 6-well plates (2 ml/well) for transfection. Day 6 supernatants were clarified and analyzed using a human IgG ELISA (ZeptoMetrix). Error bars represent the standard deviation of triplicate technical replicates.

Fig. 4 | More Antibody is Secreted on Per-cell basis with the CHOgro^® Expression System. Human IgG1 was produced by transient transfection using TransIT-PRO^® (1:1) or FreeStyle™ MAX (1.25:1.25) transfection reagents according to the manufacturers’ 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^® or FreeStyle™ System, 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. (A) Cells were analyzed using antibody capture. Briefly, an aliquot of cells was washed, incubated with an anti-IgG-PE antibody and blocking agent, washed and assayed for fluorescence. (B) Fluorescence was measured using a Guava^® easyCyte™ 5HT flow cytometer. 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.

Fig. 5 | Suspension CHO Cells Grow to High Density in the CHOgro^® Expression Medium. Triplicate flasks of FreeStyle™ CHO-S cells were seeded in CHOgro^® Expression Medium (red line) or FreeStyle™ CHO Expression Medium (blue line) at cell density of 0.5 x 10⁶ cells/ml, 40 ml per 125 ml shake flask (Thomson). Cell counts (solid line) and viability (propidium iodide staining, dotted line) were measured daily using a Guava^® easyCyte™ 5HT flow cytometer (EMD Millipore). Error bars represent the standard deviation of three readings of biological triplicates.

Fig. 6 | CHOgro^® Media Exchange Leads to Higher Protein Production. FreeStyle™ CHO-S cells were cultured in FreeStyle™ CHO Expression Medium or CHOgro^® Expression Medium. Twenty four hours prior to transfection a subset of the cells grown in FreeStyle™ CHO Expression Medium were spun down and exchanged with 100% fresh CHOgro^® Expression Medium. The cells were allowed to grow and adapt for 24 hours prior to transfection with FreeStyle™ MAX (1.25:1.25) or TransIT-PRO^® (1:1) transfection reagents according to the manufacturers’ protocol (reagent:DNA ratio) and a human IgG1 encoding construct. Transfections were performed using 1 µg plasmid DNA per milliliter of culture and cell densities of 1 x 10⁶ cells/ml for cells transfected with FreeStyle™ Max and 2 x 10⁶ cells/ml for cells transfected with TransIT-PRO^®. All cells were plated into non-treated 6-well plates (2ml/well) for transfection. (A) Workflow schematic of media exchange of CHO-S cells from FreeStyle™ CHO Expression Medium to CHOgro^® Expression Medium (black arrow) or the normal CHOgro^® Expression System (red arrow) (B) Day 6 supernatants were clarified and analyzed using a human IgG ELISA (ZeptoMetrix). Data is normalized to the complete CHOgro^® Expression System (red bar). Error bars represent the standard deviation of triplicate technical replicates.

Fig. 7 | Less Cell Clumping is Observed with the CHOgro^® Expression System. FreeStyle™ CHO-S cells were cultured in CHOgro^® Expression Medium or FreeStyle™ CHO Expression Medium and seeded into a 125 ml shake flask (20ml culture volume, Thomson) for transfection. Human IgG1 was produced by transient transfection using TransIT-PRO^® (1:1) or FreeStyle™ MAX (1.25:1.25) transfection reagents according to the manufacturers’ protocol (reagent:DNA ratio). Transfections were performed using 1 µg or 1.25 µg plasmid DNA per milliliter of culture and cell densities of 2 x 10⁶ cells/ml or 1x 10⁶ cells/ml for the CHOgro^® or FreeStyle™ System, respectively, at the time of transfection. Pictures were taken of representative flasks and cells (inset) 6 days post-transfection.

Fig. 8 | 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.

Fig. 9 | 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.