Editing 293E Protein Production (section)

=== <br>METHOD  ===

<br>Small-Scale Transfection in Six-Well Plates or 125-mL Shake Flasks 

While the protocol described here uses a DNA:PEI ratio of 1:2, the ratio selected for production should be determined by testing various ratios in six-well plate experiments. These small-scale tests must include regular samplings to determine the optimal harvest periods. The flask procedure is scalable to 650 mL in a 2-L shake flask. 

1. One or two days prior to transfection, dilute the cells to 2.5 × 105 cells/mL (for 48 h prior) or 5 × 105 cells/mL (for 24 h prior) with the appropriate cell culture medium. 

Optimal cell density should be reached on the day of transfection. This avoids having to centrifuge the cells, which can have a detrimental effect on transfection efficiency if performed shortly prior to transfection. 

2. On the day of transfection, warm the PBS to 25°-37°C. Thaw the DNA and PEI. 

3. From a maintenance flask, determine the cell density and viability using a hemocytometer. 

Cell density at transfection should range from 8 × 105 to 1.2 × 106 cells/mL, provided the doubling time is 24 h and viability is greater than 95%. 

4. Add 1.8 mL of cells to each of the six wells (or 18 mL to a new 125-mL shake flask). Transfer to an incubator. 

To minimize medium evaporation, put the plates in a plastic container lined with a wet paper towel to preserve humidity. “Trap” some CO2 in the container before closing the lid. 

5. Add 200 μL of PBS to six 1.5-mL tubes (or 2.0 mL to a 15-mL tube for the 125-mL shake flask). 

6. Add 2 μg of DNA to each tube (or 20 μg to the 15-mL tube for the 125-mL shake flask). Vortex gently. 

To evaluate transfection efficiency, add a GFP expression plasmid at 5% of the final DNA content used. A reasonable indicator of successful transfection is when the percentage of GFP-positive cells is between 30% and 50% by 48-72 h post-transfection. 

7. Add 4 μL of PEI to each tube (or 40 μL to the 15-mL tube for the shake flask) containing DNA solution. Vortex immediately (three times, 3 sec each) after PEI addition. 

Compare different DNA:PEI ratios (1:2 vs. 1:3, etc.) to see whether different lots and/or production batches affect transfection efficiency and productivity. Each new PEI preparation must be tested. 

8. Incubate the mixture for 15 min at room temperature. 

9. Remove the culture (from Step 4) from the incubator. Add the DNA/PEI mixture(s) and swirl. Return the culture to the incubator. 

10. For secreted protein production, add 50 μL of prewarmed TN1 medium at 24-48 h post-transfection to each of the six wells (or 500 μL for the 125-mL shaker flask). Return the culture to the incubator. 

The final TN1 concentration in the culture(s) should be 0.5% (w/v). TN1 supplementation is not necessary for intracellular protein production. 

11. Harvest the r-proteins: 

For intracellular r-proteins 

i. Harvest the cells at 48-72 h post-transfection. 

Establish the best harvesting time by performing an expression kinetics study. 

For secreted r-proteins 

ii. Harvest the supernatants between 96 and 168 h post-transfection as long as the culture viability is high and the product titer is increasing. 

Since culture viability decreases with time, it is important to harvest when the viability is relatively high (i.e., greater than 75%) to protect the r-protein from proteolytic degradation. 

See Troubleshooting.