Frequently Asked Questions:

CHOgro® High Yield Expression System

The CHOgro® High Yield Expression System is an advanced and cost-effective transient transfection system for high-yield protein production in suspension CHO cells. Our second-generation system incorporates the CHOgro® Titer Enhancer to provide industry leading protein yields as compared to the original CHOgro® Expression System and the ExpiCHO Expression System (ThermoFisher Scientific). The complete CHOgro® High Yield Expression System consists of CHOgro® Expression Medium, L-Glutamine and Poloxamer 188 medium supplements,TransIT-PRO® Transfection Reagent, CHOgro® Titer Enhancer and CHOgro® Complex Formation Solution.

GENERAL QUESTIONS AND ANSWERS

Q1. What components are included in the complete CHOgro® High Yield Expression System (MIR 6270)?
Q2. How should the CHOgro® High Yield Expression System components be stored?
Q3. What is the composition of the CHOgro® Expression Medium?
Q4. Does the CHOgro® Expression Medium require supplementation before use?
Q5. What is the composition of TransIT-PRO® Transfection Reagent?
Q6. What is the mechanism of action of TransIT-PRO® Transfection Reagent?
Q7. What is the composition and mechanism of action of CHOgro® Titer Enhancer?
Q8. What suspension CHO-derived cell lines can be used with the CHOgro® High Yield Expression System?
Q9. Is the CHOgro® Expression Medium suitable for CHO-GS cell selection?
Q10. Is the CHOgro® Expression Medium suitable for selection with DG44 or DUX cells?
Q11. How does the CHOgro® High Yield System compare to the ExpiCHO Expression System (Life Technologies) for antibody production?
Q12. How does the CHOgro® High Yield System compare to the FectoCHO Expression System (Polyplus) for antibody production?
Q13. What are the advantages of using CHOgro® Expression Medium over other media formulations when using the TransIT-PRO® Transfection Reagent?
Q14. What protein yield can I expect using the CHOgro® High Yield Expression System?
Q15. Are all components of the CHOgro® High Yield Expression System animal-origin-free?

PROTOCOL QUESTIONS AND ANSWERS

Q16. Which serum-free medium should I use for preparing TransIT-PRO® transfection complexes?
Q17. How long will it take to adapt my cells to CHOgro® from my current media formulation?
Q18. Will increasing the density of my cells at the time of transfection increase my protein titer?
Q19. How much TransIT-PRO® Reagent and DNA do I need to use per 1 liter of culture?
Q20. Will antibiotics interfere with my transfection?
Q21. Is a temperature shift from 37°C to 32°C 0-24 hours post-transfection necessary to achieve maximum titers?
Q22. What effect will a temperature shift 0-24 hours post-transfection have on my final protein yield and quality?
Q23. At what time point do you recommend harvesting post-transfection?
Q24. Can CHOgro® High Yield Expression System be used to generate stable cell lines?
Q25. How can I validate the CHOgro® High Yield Expression System?

TROUBLESHOOTING QUESTIONS AND ANSWERS

Q26. What can I do to further improve protein yields when using the CHOgro® High Yield Expression System?
Q27. Can I add supplements to the CHOgro® Expression Medium?

GENERAL QUESTIONS AND ANSWERS

Q1. What components are included in the complete CHOgro® High Yield Expression System (MIR 6270)?
The complete CHOgro® High Yield Expression System includes:

  • CHOgro® Expression Medium (MIR 6200, 2 x 1 L)
  • Media supplements: Poloxamer 188 (MIR 6230, 100 ml) and L-Glutamine (MIR 6240, 100 ml)
  • TransIT-PRO® Transfection Reagent (MIR 5740, 1.0 ml)
  • CHOgro® Titer Enhancer (MIR 6220, 20 ml)
  • CHOgro® Complex Formation Solution (MIR 6210, 100 ml)

Most components of the CHOgro® High Yield Expression System can be purchased individually or as part of the complete kit (MIR 6270). The CHOgro® Titer Enhancer is sold only as a component within the CHOgro® Transfection and Titer Enhancer Kit (MIR 6225) and the complete CHOgro® High Yield Expression System (MIR 6270). 

Q2. How should the CHOgro® High Yield Expression System components be stored?
Store the components as follows:

  • CHOgro® Expression Medium and CHOgro® Complex Formation Solution at 4°C, protected from light.
  • CHOgro® Titer Enhancer at 2-10°C, protected from light.
  • TransIT-PRO® Transfection Reagent 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. 

Q3. 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. CHOgro® Expression Medium is manufactured using cGMP-compliant processes in an ISO-compliant facility.

Q4. Does the CHOgro® Expression Medium require supplementation before use?
Yes. Prior to use, CHOgro® Expression Medium requires supplementation with L-Glutamine (20 ml per 1L, 4mM final concentration; MIR 6240) and Poloxamer 188 (30 ml per 1L, 0.3% final concentration; MIR 6230) for robust cell growth and viability.

Q5. 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.

Q6. 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.

Q7. What is the composition and mechanism of action of CHOgro® Titer Enhancer?
CHOgro® Titer Enhancer is a proprietary, chemically defined and animal-origin free formulation that modulates cellular processes to increase antibody production. The CHOgro® Titer Enhancer is sold only as a component within the CHOgro® Transfection and Titer Enhancer Kit (MIR 6625) and the complete CHOgro® High Yield Expression System (MIR 6270).

Q8. What suspension CHO-derived cell lines can be used with the CHOgro® High Yield Expression System?
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.

Q9. 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 4mM 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.

Q10. 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.

Q11. How does the CHOgro® High Yield System compare to the ExpiCHO Expression System (Life Technologies) for antibody production?
We typically observe similar or higher antibody titers with CHOgro® High Yield Expression System and ExpiCHO Expression Systems at Day 7 post-transfection when following manufacturer recommended protocols. (NOTE: Similar titers are observed with the CHOgro® High Yield Expression System when using either CHO-S or ExpiCHO-S cells.) For comparison data, please see the CHOgro® High Yield Expression System product webpage.

Q12. How does the CHOgro® High Yield System compare to the FectoCHO Expression System (Polyplus) for antibody production?  
We typically observe 5-fold higher protein titers using the CHOgro® High Yield Expression System versus the FectoCHO Expression Systems when comparing CHO-S cell transfections with cells adapted to the respective mediums. For comparison data, please see the CHOgro® High Yield Expression System product webpage.

Q13. 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, TransIT-PRO® Transfection Reagent and CHOgro® Enhancer enables robust cell growth and high efficiency transfection that streamlines the transient protein expression process.

Q14. What protein yield can I expect using the CHOgro® High Yield Expression System?
Protein yield is highly dependent on the intrinsic properties of the recombinant protein and vector construct design. For IgG antibody production data and product comparison, please see the CHOgro® High Yield Expression System product webpage.

Q15. Are all components of the CHOgro® High Yield Expression System animal-origin-free?
Yes. All components of the CHOgro® High Yield Expression System are free of any animal-derived sources. A Certificate of Origin stating this is available for all components of the CHOgro® High Yield System.

PROTOCOL QUESTIONS AND ANSWERS

Q16. 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.

Q17. 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, ExpiCHO 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. Longer adaptation times may also be used. Cells are typically considered fully adapted when they are doubling every 18-24 hours and ≥ 98% viably by trypan blue exclusion.

Q18. Will increasing the density of my cells at the time of transfection increase my protein titer?
We recommend transfecting suspension CHO cells (e.g. CHO-S, ExpiCHO-S) at a density of 4 x 106 cells/ml. In our experience, cell densities >4 x 106 cells/ml at the time of transfection do not yield higher titers.

Q19. 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.

Q20. 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). 

Q21. Is a temperature shift from 37°C to 32°C between 0-24 hours post-transfection necessary to achieve maximum titers?
Yes, this is strongly recommended. We typically observe at least 2-fold higher titers when placing cultures at 32°C following the addition of transfection complexes and CHOgro® Titer Enhancer. If cultures cannot be shifted, shorter incubation times of 3-5 days are generally recommended for 37°C cultures due to the potential for protein degradation at higher temperature.

Q22. What effect will a temperature shift 0-24 hours post-transfection have on my final protein yield and quality?
We typically see a 2-fold increase in titers when cultures are shifted from 37°C to 32°C between 0-24 hours post-transfection. For some targets, a >2-fold titer increase is observed. It is generally accepted that overall protein quality is superior in 32°C cultures as proteins degrade less rapidly under mildly hypothermic conditions.

Q23. 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 5-7 days are recommended due to decreased cell viabilities from depletion of culture nutrients.

Q24. Can the CHOgro® High Yield Expression System be used to generate stable cell lines?
The CHOgro® High Yield 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 Accessory Products Webpage.

Q25. How can I validate the CHOgro® High Yield Expression System?
Mirus Bio 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. Typically, titers of > 400 mg/L are obtained at Day 7 post-transfection with this control in the CHOgro® High Yield Expression System when following protocol recommendations for cell density at transfection (i.e. 4 x 106 cells/ml), complex formation, and temperature shift to 32°C after CHOgro® Titer Enhancer addition.

TROUBLESHOOTING QUESTIONS AND ANSWERS

Q26. What can I do to further improve protein yields when using CHOgro® High Yield 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 4 × 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 0-24 hours post-transfection, extending the culture incubation time (e.g. 10-14 days) may significantly increase 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, it is important to 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 2-fold increase in titers when cultures are shifted from 37°C to 32°C between 0-24 hours post-transfection. For some targets, a > 2-fold increase is observed. Overall protein quality is frequently superior in 32°C cultures as proteins degrade less rapidly under mildly hypothermic conditions.
  • 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 prior to transfection. Do not proceed with transfections if cells are not doubling daily and ≥ 98% viable by trypan blue exclusion.

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

Q27. 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 (e.g. highly cationic or anionic). 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.

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