Mammalian Expression for High-Yield Protein Production Support—Troubleshooting

Most 293 cell lines can be adapted directly from conventional serum-containing or other serum-free media into Expi293™ Expression Medium by either using the Direct Adaptation method or Sequential Adaptation method, described in the manual.

While the Expi293™ Expression Medium can support much higher cell densities, we do not recommend growing your Expi293F™ cultures beyond 5–6 x 10⁶ cells/mL, as subsequent transfection and protein expression efficiencies may be reduced. At higher densities, there is also the increased possibility of reaching the point of reduced culture viability. If your seed culture does exceed 5–6  x 10⁶ cells/mL, passage them once or twice as detailed in the manual, monitoring them for viability and growth rate. Perform a test transfection using the Protein Expression Control IgG or an expression construct of known yield to determine if cell expression performance has been impacted.

We do not recommend using OptiPRO SFM to make DNA–ExpiFectamine 293 transfection complexes. If you require an animal origin–free system, you may use FreeStyle 293 Expression Medium to make the complexes, but keep in mind that there will be a 10–20% drop in final protein yields.

The best transfection efficiencies are obtained when transfection complexes are used fresh. However, they should be stable for at least an hour. 

Please refer to the Application Note for tips on optimizing protein yield using the Expi293™ Expression System.

Please see the Application Note for tips on optimizing protein yield using the Expi293™ Expression System in 96-well microtiter plates.

Other 293 cell lines may be used with the FreeStyle™ 293 Expression System. However, they would have to be adapted to serum-free suspension culture in FreeStyle™ 293 Expression Medium and evaluated for transfection and expression.

Culture viability should be in the high 90% range for the seed stock that you are maintaining and growing for the transfections. After transfection, the viability will slowly decrease to the lower 90% to 80% range by about 3–6 days post-transfection and can drop more dramatically at 7 days or longer. This is when using our recommended protocol, which does not suggest any media changes or supplementation post-transfection.

A few parameters we recommend checking to assure high culture viability are: pH at ~7.0, no visible clumping of the cells, CO₂ levels at ~7–8% (this should be adjusted to achieve the appropriate pH), volume of culture at 40% or less of nominal flask capacity, and incubator temperature at 37°C. If you have a shaker platform setup in your incubator, the platform may generate sufficient heat to increase the temperature of the incubator. We recommend using a thermometer attached to the shaker platform. Use of baffled flasks may help, if insufficient oxygenation of the cultures is suspected. We also recommend handling and storing the culture media properly, as light exposure can result in breakdown of certain media components. If you are using FreeStyle™ 293 Expression Medium, supplementing with Pluronic® reagent is not necessary.

Note: The two main causes of clumping are 1) allowing the cells to grow to too high a density in culture, and/or 2) not providing sufficient agitation. Hence, clumping can be prevented by sufficient agitation of the culture, regular cell passage schedule, and maintenance of cells at recommended densities. If clumping is observed, it is suggested to discard the culture and start with a fresh vial of frozen cells. Vigorous vortexing for 10–30 seconds may be required at each subculture for a number of passages until the cultures grow predominantly as single cells. Additionally, at each passage, one can weed out the clumped cells by allowing the clumped cells to settle. One can increase the rpm of the spinner and also make sure to passage the culture each time before it grows to over 1.5–2 x 10⁶ cells/mL. As long as cell viability remains above 95%, the rpm of the spinner can be increased. At the time of transfection, decrease the rpm for at least the first 2 to 4 hours post-transfection to make sure the DNA–transfection reagent complexes attach well and enter into the cells. One can also add Anti-Clumping Agent (Cat. No. 0010057AE) at a dilution of 1:1000 (1:200 maximum) for the first two passages, then subculture without Anti-Clumping Agent prior to transfection. Do not add Anti-Clumping Agent at any time during the transfection because it will interfere with the transfection. However, Anti-Clumping Agent can be added to the cultures several hours post-transfection.

You can visually tell if MembranePro™ particles formed via a pellet at the bottom of the tube following precipitation. You can also test function of your MembranePro™ particles via receptor-ligand binding studies. 

While other cell lines can be tested, we recommend using the Expi293F™ cells, as the performance of the kit has been optimized for use with this cell line (i.e., transfection efficiency using ExpiFectamine™ Transfection Reagent). 

While other expression vectors and promoters can be tested, we recommend using the pEF6 V5-His TOPO® TA expression vector for optimal yields, as the performance of the kit has been optimized for use with this expression vector containing the EF-1α promoter. 

Some mild cell clumping is normal during ExpiCHO-S™ cell passaging and is okay as long as the cells have >95% viability. At each passage you may allow the clumps to settle to the bottom by letting the flask rest for 30 sec  to 1 min, then passage the suspended cells. If the cells have a lot of clumping paired with viability < 95%, this indicates a problem either with the culture conditions or the cells themselves.  We recommend verifying that the cells are: cultured according to the recommendations in the manual, below passage 20, and free of contamination. Thaw a new vial of cells as necessary.

Yield can vary greatly from protein to protein. We strongly recommend expressing the rabbit antibody positive control (Cat. No. A14662) to determine if the low yield is due to a low expressing protein, a problem with the system components, or transfection and culturing conditions. 

If you are not achieving the expected yield with the rabbit IgG positive control, we recommend checking the following: 

• Ensure that cells are >95% viable during normal passaging and at time of transfection.
• Have a doubling time of approximately 17 h.
• Recover cells rapidly post-thaw (within 3-4 days post thaw); if not, verify you are using the culturing guidelines provided in the manual and thaw a new vial of cells if necessary.  
• We recommend gentle swirling at all handling steps for ExpiCHO-S™ cells (avoid vigorous swirling, shaking, and pipetting up and down). Verify that your shake speed is about 110-125 rpm for 25 mm throw shakers and about 125 for 19 mm throw shakers. Test a flask with containing water and a thermometer to determine whether the shaker is putting off excess heat; reduce the incubator temperature setting as necessary to achieve a final temperature of 37 degrees C for your cultures.  All of our shake speed recommendations are provided for Corning™ non-baffled flasks; if you are using a different culture vessel, additional shake speed optimization may be required.

The optimal complexation time is 5 minutes. We have observed a small drop in protein yield (~20% reduction) if complexes sit up to 10 minutes; for 20 minutes or longer yield will be drastically reduced (>50% reduction in yield).

This drop in viability tends to indicate that some aspect of the cell culture conditions are not optimal during the expression run. If this is observed, the shaking speed of the flasks should be verified to be within protocol specifications; the volume and size of the flask should be appropriate; and care should be taken when handling the ExpiCHO-S Cells ahead of the expression run to ensure that cells are not stressed by vigorous mixing, etc.

In some instances, flask-to-flask variability has been observed using the high- and max-titer protocols at the 125 mL flask scale. Here, it is recommended to increase the shaking speed to 130 rpm for 25 mm shakers and 140 rpm for 19 mm shakers.

Alternatively, we have found that baffled flasks work well for the 125 mL scale to correct any flask-to-flask variability&mdash;in such an instance, to account for the baffles, the volume of cells to be transfected should be increased from 25 mL to 35-40 mL to allow for optimal flow over the baffles, and the volumes of other reagents should be scaled accordingly.

Lower transfection volumes (i.e., 30-35 mL) can also be used with baffled flasks; however, shaking speeds must be reduced slightly to account for the baffles (i.e., 115 rpm for 19 mm shakers and 110 rpm for 25 mm shakers). We have not found baffled flasks to be necessary or beneficial at other flask sizes.

Because the ExpiCHO system uses components that differ from that of HEK 293-based expression systems such as the Gibco Expi293 system, the supernatant may be more difficult to process through standard bottle top filters in some instances. To remedy this, we recommend centrifuging the supernatant first at ~5,000 x g for 30 minutes followed by filtration using a 0.22 µm filter. Alternative filters (such as depth filters) provide superior filtration for supernatants, especially at larger scales, as these filters are specifically designed to handle supernatant from CHO-derived expressions.

Expi293 Met (-) Expression Medium does not contain methionine, which is an essential nutrient for cell culture. Therefore, methionine supplementation is required. For methionine labeling, cells should be grown in Expi293 Met (-) Expression Medium and starved (i.e., grown in the absence of methionine supplementation) for 6 hr prior to addition of 13C-methyl-L-methionine, or similar methionine isotope.

L-selenomethionine is a known cell toxin. Therefore, it is important to grow cells in the present of this amino acid for the shortest amount of time required to achieve maximal labeling. We recommend adding L-selenomethionine to the culture just prior to transfection (in Expi293 Met (-) Expression Medium). You may also starve cells for 6 hr by culturing cells in Expi293 Met (-) Expression Medium (without supplementation) and then proceed with L-selenomethionine add-back and transfection.