Protein Production

Achieve High Antibody Titers in Suspension CHO Cells

• High Protein Yield – Better yield than PEI or FreeStyle™ MAX Transfection Reagent
• Compatible with multiple CHO media formulations – Use with CHOgro® Expression Medium to Achieve Highest Yields
• Simple Workflow – Reproducible Protein Expression with Minimal Optimization

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.

FreeStyle™ CHO-S and ExpiCHO Cells Grown in CHOgro® Expression Medium Yield Similar Titers. FreeStyle™ CHO-S cells (ThermoFisher Scientific) or ExpiCHO-S cells (ThermoFisher Scientific) were cultured in CHOgro® Expression Medium (Mirus Bio). Both cell lines were transiently transfected with plasmid DNA encoding a human IgG1 internal control antibody, Bevacizumab or Fc-fusion construct 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 in a non-treated 6-well plate. All cultures were shifted to 32°C, shaking, immediately post-addition of the transfection complexes and the CHOgro® Titer Enhancer to the culture. 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). 

CHOgro® High Yield Expression System Enables 1000-fold Scalability. Human IgG1 was produced by transient transfection with the TransIT-PRO® Transfection Reagent (Mirus Bio) and 1 µg plasmid DNA per milliliter of culture at a 1:1 reagent:DNA ratio. Freestyle CHO cells (Thermo Fisher Scientific) were transfected at a density of 4 x 10⁶ cells/ml in CHOgro® Expression Medium (Mirus Bio) at the following volumes/culture vessels: 2 ml/non-tissue culture treated 6-well dish, 20 ml/125 ml Thomson flask, 500 ml/1.6 L Thomson flask, 2000 ml/5 L Thomson flask. All cultures were shifted to 32°C, shaking, immediately post-addition of the transfection complexes and the CHOgro® Titer Enhancer to the culture. Cultures were maintained at the appropriate shake speed for the remainder of the experiment. Day 14 supernatants were analyzed using a standard sandwich human IgG ELISA. Error bars represent the standard deviation of triplicate technical replicates.

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.

Higher Titers Achieved by Third Party with CHOgro® High Yield Expression System Plus CHOgro® Titer Enhancer. Fc-fusion proteins were produced by transient transfection of CHO-S cells in complete CHOgro® Media using TransIT-PRO® Transfection Reagent (1:1 reagent:DNA ratio, volume: weight). Cells were either 2 x 10⁶ cells/ml (no CHOgro® Titer Enhancer) or 4 x 10⁶ (with CHOgro® Titer Enhancer) for transfection. Cultures were shifted to 32°C either 24 hours (no CHOgro® Titer Enhancer) or immediately (with CHOgro® Titer Enhancer) following transfection. Yields represent final affinity-purified protein from 0.5 L culture, harvested at day 11 post-transfection.

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.

Increase Expression of Secreted Proteins and Antibodies in 293 Cells

• Better or Equal Expression than 293fectin™ Transfection Reagent
• Express Secreted Proteins and Antibodies at High Levels

Achieve High Protein Yields Using TransIT-PRO® Transfection Kit in Suspension 293 Cells. Ten different secreted (non‐antibody) proteins were transiently expressed in FreeStyle™ 293‐F cells (Life Technologies) using TransIT‐PRO (1.5:1) or 293fectin™ (Life Technologies, 2:1) transfection reagents according to manufacturer’s protocol. Cells were grown in FreeStyle™ 293 Expression Medium at transfected at a density of 1 x 10⁶ cells/ml. The scale of the transfection for each protein varied between 1‐6 L of culture. More experimental details here. (PDF)

Data courtesy of a TransIT‐PRO pharmaceutical customer.

High Levels of Antibody Expression at Different Volume Scales using TransIT-PRO® Transfection Kit

TransIT-PRO® Use in the Expi293™ Expression System

• Compatible with high density transfection of Expi293F™ cells
• Streamlined protocol (below) with no enhancer addition required

Protocol for Transfecting Expi293F™ Cells using TransIT-PRO® Reagent

1. Determine the total number of cells required for your experiment at least one day prior to transfection. Each 30 ml transfection requires 7.5 × 10⁷ cells in 25.5 ml of Expi293™ Expression Medium.
2. Seed the cells at a density of 2.0 × 10⁶ viable cells/ml and maintain 18 – 24 hours prior to transfection to ensure that cells are actively dividing at the time of transfection. Shake flasks overnight on an orbital shaker (125 rpm when using a shaker with a 2 cm orbital throw) at appropriate temperature and CO₂ levels (e.g. 37°C, 8% CO₂).
3. On the day of transfection, quantitate cell number and viability using an automated cell counter or using the trypan blue dye exclusion method. Do not proceed with transfection unless cells are >95% viable.
4. Determine the volume of cell suspension containing the number of cells needed for one transfection. Each 30ml transfection requires 7.5 × 10⁷ cells (i.e. 2.5 × 10⁶ cells/ml final concentration).
5. Add the appropriate volume of cell suspension to each sterile, disposable 125 ml Erlenmeyer shake flask and bring up the volume to 27 ml by adding fresh, pre-warmed Expi293™ Expression Medium for each 30 ml transfection. Return the cells to the incubator.
6. For each 30 ml transfection, prepare reagent-DNA complexes as follows:
   • In a sterile tube, dilute 30 μg of plasmid DNA in Opti-MEM® I Reduced Serum Medium (Life Technologies) to a total volume of 3 ml. Mix gently but thoroughly.
   • Add 30 μl of TransIT-PRO® Transfection Reagent to the tube containing diluted DNA. Mix gently but thoroughly.
   • Incubate the DNA and TransIT-PRO® Transfection Reagent mixture for 20–30 minutes at room temperature to allow transfection complexes to form.
   NOTE: This procedure recommends a 1:1 TransIT-PRO®:DNA ratio, which results in the optimal titers for most proteins. However, customer feedback indicates that a 2:2 ratio (i.e. 60 µl TransIT-PRO® + 60 µg DNA per 30 ml culture volume) may result in improved protein expression with some constructs.
7. After the DNA-TransIT-PRO® Transfection Reagent complex incubation is complete, add 3 ml of DNA-TransIT-PRO® Transfection Reagent complex alone to each shaker flask from Step 5. To the negative control flask, add 3 ml of Opti-MEM® I medium instead of DNA-TransIT-PRO® Transfection Reagent complex. Each flask should contain a total volume of 30 ml.
   NOTE: No enhancer addition is required with the TransIT-PRO® Reagent.
8. Shake flasks on an orbital shaker (125 rpm when using a shaker with a 2 cm orbital throw) at appropriate temperature and CO₂ levels (e.g. 37°C, 8% CO₂).
9. Cells or media (if recombinant protein is secreted) may be harvested beginning at approximately 48 hours post-transfection and assayed for recombinant protein expression. The incubation time for optimal protein expression depends on the nature of the recombinant protein.

Expi293™ and Expi293F™ are trademarks of Life Technologies Corporation.

Download Protocol: TransIT-PRO® Transfection Reagent for EXPI293F™ Cells (PDF)