TransIT-PRO® Transfection Kit

Figure 1. Achieve High Antibody Titers Using the TransIT-PRO® Transfection Kit in Suspension CHO Cells. Human IgG1 was produced by transient transfection using TransIT-PRO and PRO Boost Reagent (1:1:1), 25 kDa linear PEI (6:1) or FreeStyle™ MAX (1:1) 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 0.5 x 10⁶ cells/ml at the time of transfection. FreeStyle™ CHO-S cells were cultured in 20 ml of FreeStyle™ CHO Expression medium in 125 ml shake flasks. (A) Day 3, 5 and 7 supernatants were clarified and analyzed using a human IgG-Fc sandwich ELISA. Error bars represent the standard deviation of triplicate technical replicates, 25kDa linear PEI is duplicate technical replicates. (B) Day 7 supernatants were clarified and analyzed by Western blot. An IgG standard was included for quantification estimate (S1= 1.6 mg/L, S2= 3.2 mg/L, S3 = 6.3 mg/L).

Figure 2. TransIT-PRO® Provides High Performance Across Varied Media Formulations. FreeStyle™ CHO-S cells were adapted to five representative growth media including: BD Select™ CD1000 medium (Becton, Dickinson and Company, Franklin Lakes, NJ), BD Select™ CHO medium (Becton, Dickinson and Company, Franklin Lakes, NJ), FreeStyle™ CHO Expression medium (Life Technologies Corporation, Carlsbad, CA), ProCHO™5 medium (Lonza Inc., Allendale, NJ) and PowerCHO^®2 CD medium (Lonza Inc., Allendale, NJ). Cells were transfected with a plasmid using the TransIT-PRO and PRO Boost Reagent (1:1:1), 25 kDa linear PEI (4:1) (Polysciences, Warrington, PA), or FreeStyle™ MAX (1:1) (Life Technologies Corporation, Carlsbad, CA) transfection reagents according to the manufacturers or published protocol (reagent:DNA ratio). Transfections were performed in 24-well deep well shaker blocks using 1 µg plasmid DNA per milliliter of culture and 0.5 x 10⁶ cells/ml at the time of transfection. (A)  Luciferase expression was assessed 48 hours post-transfection using a conventional luciferase assay. Error bars represent the standard deviation of duplicate wells. (B) Human IgG1 was quantitated from day 5 clarified supernatants and analyzed by a human anti-Fc sandwich ELISA. Error bars represent the standard deviation of triplicate wells.

Figure 3. Scaling of Transient Transfection Using TransIT-PRO® Transfection Kit is Linear From 4 to 400 Milliliters. Human IgG1 was produced by transient transfection using the TransIT-PRO Transfection Kit and a plasmid encoding a human IgG1 construct. A DNA concentration was 1µg/ml of culture and a ratio of TransIT-PRO:PRO Boost Reagent:DNA ratio of 1:0.5:1. Cells were plated at a density of 0.5 x 10⁶ cells/ml at the time of transfection. CHO-S cells were cultured in BD Select™ CD1000 media using 4 ml per well of a 24-well deep well shake block, 40 ml in 125 ml Erlenmeyer shake flask, 40 ml in 125 ml 2 sidearm spinner flask and 400 ml in 500 ml 2 sidearm spinner flask. Day 3, 5 and 7 supernatants were clarified and analyzed by an anti-Fc sandwich ELISA. Error bars represent the standard deviation of triplicate technical replicates.