Editing 293E Protein Production (section)

=== <br><br>METHOD  ===

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Although the protocol described here uses a DNA:PEI ratio of 1.5:2, 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. 

1. Warm the LC-SFM culture medium to 37°C. 

2. Remove the cell maintenance T-flask from the incubator. Dislodge the cells by gently tapping the side of the flask 

This is not necessary if the maintenance cultures are in shake flasks. 

3. Remove a cell aliquot to determine cell density and viability using a hemocytometer. 

4. In a 15-mL tube, dilute the cells to 2.5 × 105 cells/mL in a volume of 12 mL of medium. 

This can be scaled down to 1 mL in 12-well plates. 

5. Add 1.8 mL of cells to each of the six wells. Transfer to the incubator. Culture for 24 h. 

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. 

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

7. Aliquot 100 μL of PBS to each of six 1.5-mL tubes. 

8. Add 3 μg of DNA to each tube. Vortex. 

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. 

9. To another 1.5-mL tube, add 672 μL of PBS. Add 28 μL of branched PEI solution. Vortex. 

10. Add 100 μL of PEI solution to each DNA-containing tube. Vortex immediately (three times, 3 sec each) after PEI addition. 

Compare different DNA:PEI ratios to see whether different lots and/or production batches affect transfection efficiency and productivity. Each new PEI preparation must be tested. 

11. Incubate for 15 min at room temperature. 

12. Remove the six-well plate from the incubator. Add the DNA/PEI mixture to each well and swirl the plate. Return the plate to the incubator. 

13. For secreted protein production, add 50 μL of prewarmed TN1 medium at 24-48 h post-transfection to each of the six wells. 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. 

14. 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. 

Because 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. 

<br>TROUBLESHOOTING 

Problem: The transfection does not work. 

[Step 14] 

Solution: Consider the following: 

1. Make sure the cells are in exponential growth phase for transfections. If the culture grows to higher-than-prescribed transfection densities, medium nutrient depletion and metabolic by-products will compromise the transfection. 

2. Anti-clumping agents such as dextran sulfate can inhibit transfection. 

3. DNA plasmid quality may be inadequate. Check for contaminants by A260/A280 ratio readings; acceptable values range between 1.80 and 1.95. In addition, the DNA plasmid can be digested and run on an agarose gel to verify its integrity (it should be mostly in a supercoiled form) and/or to detect RNA contamination. 

4. Monitor cell line productivity occasionally by transfecting the cells with an easily measured protein such as secreted alkaline phosphatase or GFP. Low transfection efficiencies are readily indicated when the percentage of GFP-positive cells is below 30% by 48 h post-transfection. Also, for each new medium or reagent lot, monitor transfection efficiencies using six-well plate assays. 

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