Achieve High Antibody Titers in Suspension CHO Cells

The CHOgro® High Yield Expression System Outperforms the ExpiCHO Expression System Using Multiple Antibody Constructs. Six different IgG1 antibody constructs, including five therapeutically relevant constructs synthesized in the same vector backbone were produced by transient transfection using either the TransIT-PRO® Transfection Reagent (Mirus Bio) at a 1:1 reagent-to-DNA ratio (vol:wt) and 1 µg plasmid DNA per milliliter of culture in FreeStyle™ CHO-S cells (ThermoFisher Scientific) cultured in CHOgro® Expression Medium (Mirus Bio) at a cell density of 4 x 10⁶ cells/ml, or the Expifectamine CHO Transfection Kit (ThermoFisher Scientific) at a 3.2:1 reagent-to-DNA (vol:wt) and 1 µg plasmid DNA per milliliter of culture in ExpiCHO-S cells (ThermoFisher Scientific) cultured in ExpiCHO Expression Medium (ThermoFisher Scientific) at a cell density of 6 x 10⁶ cells/ml. All cells were split into 125 ml Optimum Growth flask (25 ml/flask, Thomson Instrument Company) at the time of transfection. The CHOgro® Titer Enhancer was added immediately post-addition of transfection complexes and the culture was incubated at 32°C shaking until harvest for the CHOgro® High Yield Expression System. The Max titer protocol was followed for the ExpiCHO Expression System (ThermoFisher Scientific): Expifectamine CHO Enhancer and Feed (ThermoFisher Scientific) were added at 24 hours post-transfection and cultures were shifted to 32°C. At Day 5 a second volume of the ExpiCHO Feed (ThermoFisher Scientific) was added to the flask. Day 7 and 14 supernatants were analyzed using a standard sandwich human IgG ELISA. Error bars represent the standard deviation of triplicate technical replicates.

Validation of Increased Titers Obtained with CHOgro® Titer Enhancer. An Fc-fusion protein was produced by transient transfection of CHO-S cells in complete CHOgro® Medium (Mirus Bio) using TransIT-PRO® Transfection Reagent (Mirus Bio) at a 1:1 reagent-to-DNA ratio (vol:wt). As indicated, CHOgro® Titer Enhancer (Mirus Bio) was added and the culture was shifted to 32˚C following transfection. Yields represent final affinity-purified protein from 0.5 L culture, harvested at day 10 post-transfection. Data courtesy of Lytic Solutions, LLC.

Titer Enhancement is Observed Using Multiple Fc-fusion Constructs. Two different Fc-fusion constructs were transiently transfected using the TransIT-PRO® Transfection Reagent (Mirus Bio) at a 1:1 reagent-to-DNA ratio (vol:wt) and 1 µg plasmid DNA per milliliter of culture. FreeStyle™ CHO-S (ThermoFisher Scientific) cells were cultured in CHOgro® Expression Medium (Mirus Bio) and plated at 4 x 10⁶ cells/ml at the time of transfection in non-treated 6-well plates (2 ml/well). As indicated, CHOgro® Titer Enhancer was added and the culture was shifted to 32°C immediately post-addition of the transfection complexes to the culture. Day 10 supernatants were analyzed using a standard sandwich human IgG ELISA. Error bars represent the standard deviation of triplicate technical replicates.

TransIT-PRO® + CHOgro® Titer Enhancer Outperforms PEImax and linear 25 kDa PEI Transfection Reagents. An IgG1 antibody construct was produced by transient transfection using either the TransIT-PRO® Transfection Reagent (Mirus Bio) at a 1:1 reagent-to-DNA ratio (vol:wt), PEImax (4:1, Polysciences), or linear 25 kDa PEI (6:1, Polysciences) and 1 µg plasmid DNA per milliliter of culture in FreeStyle™ CHO-S cells (ThermoFisher Scientific) cultured in CHOgro® Expression Medium (Mirus Bio) at a cell density of 4 x 10⁶ cells/ml. CHOgro® Titer Enhancer (Mirus Bio) was added immediately post-addition of transfection complexes and cultures were incubated at 32°C with shaking until harvest for all reagents. Day 14 supernatants were analyzed using a standard sandwich human IgG ELISA. Error bars represent the standard deviation of triplicate technical replicates.

CHOgro® Titer Enhancer Does Not Adversely Affect Cell Growth and Viability Post-transfection. Triplicate flasks of FreeStyle™ CHO-S cells adapted to CHOgro® Expression Medium were transiently transfected with the TransIT-PRO® Transfection Reagent (Mirus Bio) at a 1:1 reagent-to-DNA ratio (vol:wt) and 1 µg plasmid DNA per milliliter of culture at 4 x 10⁶ cells/ml in 125 ml Optimum Growth flasks (25 ml/flask, Thomson Instrument Company). As indicated, CHOgro® Titer Enhancer was added and all of the cultures were shifted to 32°C immediately post-addition of the transfection complexes to the culture. Cell counts (solid line) and viability (propidium iodide staining, dotted line) were measured daily post-transfection using a Guava® easyCyte™ 5HT flow cytometer (EMD Millipore). 

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.