Frequently Asked Questions:

CHOgro® Expression System

The CHOgro® Expression System was developed through systematic optimization of transfection protocol parameters including: cell density, transfection reagent, media formulation and culture temperature. This system consists of CHOgro® Expression Medium, L-Glutamine and Poloxamer 188 medium supplements, TransIT-PRO® Transfection Reagent, and CHOgro® Complex Formation Solution. With the CHOgro® Expression System, high protein titers can now be achieved in suspension CHO cells through high density transient transfection. See also: CHOgro® High Yield Expression System.

GENERAL QUESTIONS AND ANSWERS

Q1. What components are included in the complete CHOgro® Expression System?
The complete CHOgro® Expression System includes CHOgro® Expression Medium (MIR 6200, 2 x 1 L), TransIT-PRO® Transfection Reagent (MIR 5740, 1.0 ml), CHOgro® Complex Formation Solution (MIR 6210, 100 ml), and media supplements Poloxamer 188 (MIR 6230, 100 ml) and L-Glutamine (MIR 6240, 100 ml). Components of the CHOgro® Expression System can be purchased individually or as part of the complete kit (MIR 6260).

Q2. What is the composition of the CHOgro® Expression Medium?
CHOgro® Expression Medium is a proprietary formulation that is chemically defined, hydrolysate-free and animal-origin-free.

Q3. What is the composition of TransIT-PRO® Transfection Reagent?
The TransIT-PRO® Transfection Reagent is a proprietary, animal-origin-free, lipid-polymer mixture developed for high transfection performance in suspension CHO and HEK 293 cell types.

Q4. What is the mechanism of action of TransIT-PRO® Transfection Reagent?
The TransIT-PRO® Reagent complexes with negatively charged DNA to form lipopolyplexes that associate with the negatively charged cell membrane via electrostatic interactions. These lipopolyplexes are taken into the cell via endocytosis.

Q5. Can the CHOgro® Expression System be used with other suspension CHO derived lines?
At Mirus, we have tested adaptations of the FreeStyle™ CHO-S and ExpiCHO-S Cells (Life Technologies) as well as suspension adapted CHO-K1 cells. We have received positive feedback from customers testing CHOK1SV and CHOZN-GS cells with CHOgro® Expression Medium.

Q6. Is the CHOgro® Expression Medium suitable for CHO-GS cell selection?
Yes. The CHOgro® Medium does not contain L-Glutamine but we typically recommend supplementation with 4 mM L-Glutamine (final concentration) prior to use. For CHO-GS selection, omit L-Glutamine from the culture media following transfection with plasmid DNA containing an expression cassette for glutamine synthetase.

Q7. Is the CHOgro® Expression Medium suitable for selection with DG44 or DUX cells?
No. Researchers will not be able to do selection with DG44 or DUX cells with CHOgro® Medium as it contains hypoxanthine and thymidine.

Q8. How does the CHOgro® Expression System compare to the FreeStyle™ MAX CHO System (Life Technologies)?
We observe significantly higher protein yields using the CHOgro® Expression System versus the FreeStyle™ MAX System when comparing CHO-S cell transfections with cells adapted to the respective mediums. For product comparisons, please see the CHOgro® Expression System product webpage.

Q9. What are the advantages of using CHOgro® Expression Medium over other media formulations when using the TransIT-PRO® Transfection Reagent?
CHOgro® Expression Medium is a chemically defined, nutrient rich, serum-free growth medium that permits high density growth and large-scale transfection of suspension CHO cells. Suspension CHO cells readily adapt to CHOgro® Expression Medium, making the adaptation process often required with other systems a non-issue. The combination of CHOgro® Expression Medium and TransIT-PRO® Transfection Reagent enables robust cell growth and high efficiency transfection that streamlines the transient protein expression process.

Q10. What protein yield can I expect using the CHOgro® Expression System?
Protein yield is highly dependent on the intrinsic properties of the recombinant protein. With that in mind, our CHOgro® Expression System generally yields 5X more antibody than other existing CHO cell expression systems (e.g. Freestyle MAX CHO, Life Technologies). For product comparisons, please see the CHOgro® Expression System product webpage.

Q11. How should the CHOgro® Expression System components be stored?
Store the components as follows:

CHOgro® Expression Medium (MIR 6200) and CHOgro® Complex Formation Solution (MIR 6210) at 4°C, protected from light.
TransIT-PRO® Transfection Reagent (MIR 5740) at –20°C, but warm to room temperature and vortex gently before each use.
Poloxamer 188, 10% Solution (MIR 6230) at room temperature.
L-Glutamine (MIR 6240) at –20°C and avoid multiple freeze/thaw cycles.

PROTOCOL QUESTIONS AND ANSWERS

Q12. Which serum-free medium should I use for preparing TransIT-PRO® transfection complexes?
For best transfection results in suspension CHO cells, we recommend using CHOgro® Complex Formation Solution with TransIT-PRO® Transfection Reagent. Some serum-free media formulations contain polyanions such as heparin, or polydextran sulfate that may inhibit formation of active transfection complexes.

Q13. How long will it take to adapt my cells to CHOgro® from my current media formulation?
Suspension CHO cells grown in alternate media formulations (e.g. FreeStyle™ CHO Expression Medium) can be centrifuged (300 x g for 5 minutes) 18-24 hours prior to seeding for transfection and resuspended in 100% CHOgro® Expression Medium supplemented with 4mM L-Glutamine and 0.3% Poloxamer 188.

Q14. Will increasing the density of my cells at the time of transfection increase my protein titer?
In our experience, cell densities >2 x 10⁶ cells/ml at the time of transfection will have a minimal effect on increasing titer.

Q15. How much TransIT-PRO® Reagent and DNA do I need to use per 1 liter of culture?
As a starting point, we recommend a TransIT-PRO® Reagent:DNA ratio of 1:1 (1 µl of TransIT-PRO® reagent and 1 µg of DNA per mL of culture). Therefore, 1 L of culture would require 1 mL of the TransIT-PRO® Transfection Reagent.

Q16. Will antibiotics interfere with my transfection?
Some antibiotics are cationic and will inhibit transfection complex formation. We recommend excluding antibiotics from the complex formation step. However, once the transfection complexes are formed, they can be added to cells grown in complete culture medium containing low levels of antibiotics (e.g. 100X stock of penicillin/streptomycin diluted up to 0.1-1X final concentration).

Q17. Is a media change required post-transfection?
No. A media change is not required following transfection with the CHOgro® Expression System.

Q18. Is a temperature shift from 37°C to 32°C 24 hours post-transfection necessary?
No. However, shorter incubation times of 3-5 days are generally recommended for 37°C cultures due to the potential for protein degradation at higher temperature.

Q19. What effect will a temperature shift 24 hours post-transfection have on my final protein yield and quality?
We typically see a boost in titer of at least 25% when cultures are shifted from 37°C to 32°C at a time point of 24 hours post-transfection. For some targets, a ≥ 2-fold titer increase is observed. Overall protein quality is frequently superior in 32°C cultures as proteins degrade less rapidly under mildly hypothermic conditions.

Q20. At what time point do you recommend harvesting post-transfection?
We recommend that you determine the optimal transfection incubation parameters for each cell type and experiment. The optimal incubation time will vary depending on the goal of the experiment and the nature of the plasmid used. For secreted antibody constructs, optimal titers are typically obtained at 32°C, 7‒14 days post-transfection in batch culture. For 37°C cultures, shorter incubation times of 3-5 days are recommended due to the potential for protein degradation at higher temperature.

Q21. Can the CHOgro® Expression System be used to generate stable cell lines?
The CHOgro® Expression System was designed for high titer, transient protein production in suspension CHO cell types, but it is also suitable for generating stable cell lines. For a list of selection antibiotics available through Mirus Bio, please visit our accessories webpage.

Q22. How can I validate the CHOgro® Expression System?
Mirus Bio also sells a Human IgG1 Expression Control (MIR 6250) which contains a mixture of codon-optimized, CMV-driven plasmids that express the heavy and light chains (at a 1:1 molar ratio) of a human IgG1 secreted antibody. Titers of > 150 mg/L are obtained with these constructs in the CHOgro® Expression System. The Human IgG1 Expression Control incorporates DNA2.0 IP-Free© vector technology.

TROUBLESHOOTING QUESTIONS AND ANSWERS

Q23. What can I do to further improve protein yields when using the CHOgro® Expression System?
Multiple factors can contribute to transfection efficiency and ultimate yield; refer to the following critical troubleshooting tips to improve your transfection results:

Cell density (% confluence) not optimal at time of transfection - Typically, a cell density of 2 × 10⁶ cells/ml at the time of transfection is desired, though some applications may require lower or higher densities. Ideally, cells should be split 18‒24 hours prior to transfection to ensure active cell division.
Suboptimal TransIT-PRO® Reagent to DNA ratio - Determine the optimal TransIT-PRO® Reagent:DNA ratio by titrating the reagent from 0.5-2 µl per 1 µg DNA.
Poor quality of DNA - DNA used for transfection should be highly purified, sterile, and free from contaminants such as endotoxin. Remove any traces of endotoxin (bacterial lipopolysaccharide, LPS) using MiraCLEAN® Endotoxin Removal Kit (MIR 5910 or 5900).
Culture time post-transfection – For cultures shifted to 32°C at 24 hours post-transfection, extending the culture incubation time (e.g. 10-14 days) may greatly increase your titer without impacting the quality of the protein. For antibodies or proteins liable to degradation, shorter time points (e.g. 5-7 days) may be required. Ultimately, we recommend that you evaluate your system prior to scale up to determine the optimal expression parameters.
Shifting cells from 37°C to 32°C 24 hours post-transfection – We typically see a boost in titer of at least 25% when cultures are shifted from 37°C to 32°C 24 hours post-transfection. For some targets, a ≥ 2-fold increase is possible.
Shake/spin culture conditions not optimal – Excessive agitation is harmful to cells. We recommend that you incubate cells in a shake flask at an appropriate rpm (e.g. 125 rpm with a 1.9 cm orbital throw) at 37°C, 8% CO2.

For additional tips, please refer to the Troubleshooting Guide in the user protocol.

Q24. Can I add supplements to the CHOgro® Expression Medium?
Yes. However, we recommend testing your transfection conditions with and without the media supplement to ensure that it does not inhibit transfection. If a supplement that is inhibitory to transfection is needed for long-term growth and increased protein yields, it can be added to the media 4 hours post-transfection.

Frequently Asked Questions:

CHOgro® Expression System

GENERAL QUESTIONS AND ANSWERS

PROTOCOL QUESTIONS AND ANSWERS

TROUBLESHOOTING QUESTIONS AND ANSWERS

GENERAL QUESTIONS AND ANSWERS

PROTOCOL QUESTIONS AND ANSWERS

TROUBLESHOOTING QUESTIONS AND ANSWERS

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