Frequently Asked Questions:

TransIT-PRO® Transfection Kit

TransIT-PRO® Transfection Kit consists of a DNA transfection reagent and boost combination specifically developed for mammalian protein production in suspension 293 and CHO derived cells. The PRO Boost Reagent is optional and enhances expression in certain media formulations. TransIT-PRO® Transfection Reagent and PRO Boost Reagent are comprised of animal-origin free components that are compatible with many chemically defined media formulations. Use of TransIT-PRO® Transfection Kit eliminates the need for a culture medium change post-transfection and is suitable for both transient and stable transfection.

GENERAL QUESTIONS AND ANSWERS

Q1. What is the composition of TransIT-PRO® Transfection Kit?
TransIT-PRO® Transfection Kit consists of two animal origin-free components - a DNA transfection reagent called TransIT-PRO® and an optional PRO Boost Reagent. The TransIT-PRO® Reagent is a proprietary lipid-polymer mixture and the PRO Boost Reagent is an organic compound.

Q2. What is the mechanism of action of TransIT-PRO® Transfection Kit?
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 thought to be taken into the cell via endocytosis. PRO Boost is a transfection booster identified during an empirical screening of Mirus compounds. However, the mechanism of the PRO Boost reagent is not well understood but appears to be medium-dependent. PRO Boost works best when added directly to the complex, suggesting a role in complex formation or protection of the complexes during the entry process.

Q3. Can TransIT-PRO® Transfection Kit be used to transfect other suspension CHO derived lines (e.g. CHO-DG44 cells etc.)?
At Mirus, we have only tested adaptations of the FreeStyle™ CHO-S cells from Life Technologies. We have received positive feedback from customers testing the TransIT-PRO® Transfection Kit on other CHO-S derived cells including the DG44 lines.

Q4. How does TransIT-PRO® compare to the FreeStyle™ System from Life Technologies?
We observe equivalent or higher protein yields using TransIT-PRO® versus FreeStyle™ Max Transfection Reagent to transfect FreeStyle™ CHO-S cells adapted to FreeStyle™ CHO Expression medium. Additionally, TransIT-PRO® exhibits broader media compatibility compared to the FreeStyle™ Max reagent. For detailed data, please see the TransIT-PRO® product webpage.

Q5. What is the effect on cellular viability following transfection with TransIT-PRO® Transfection Reagent?
Similar to all transfection methods, cells divide less rapidly following transfection with TransIT-PRO® compared to non-transfected cells. Initial comparison studies with leading competitors suggest that lower cell densities are obtained following transfection with TransIT-PRO®; however, protein titers are equal or higher. This reduction in cell mass, but not protein yield, might be beneficial to many researchers during downstream processing.

Q6. What kind of protein yield (mg/L) can I expect using the TransIT-PRO® Transfection Kit?
Protein yield is highly dependent on the intrinsic properties of the recombinant protein making it impossible to predict the protein yields of a particular construct. Furthermore, the nature of the suspension adapted cell types as well as the media formulation can have a huge impact on the final protein yield. Amongst antibody constructs that constitute a big fraction of biotherapeutic proteins, some subtypes (e.g. IgG1) express at higher levels. Ultimately, it is best to perform a side-by-side comparison with your current transfection method.

PROTOCOL QUESTIONS AND ANSWERS

Q7. How should TransIT-PRO® Transfection Kit be stored?
Both the TransIT-PRO® Transfection Reagent and PRO Boost Reagent should be stored at -20°C.

Q8. Which serum-free medium should I use for preparing TransIT-PRO®: PRO Boost:DNA transfection complexes?
For best results, we recommend using Opti-PRO™ Serum Free Medium (Opti-PRO™ SFM) for preparing transfection complexes using the TransIT-PRO® Transfection Kit. Some serum free media contain polyanions such as heparin, or polydextran sulfate that may inhibit formation of active transfection complexes. If it is necessary to use media containing polyanions for your experimental set-up, the transfection medium can be replaced with polyanion containing medium 24 hours post transfection.

Q9. How much TransIT-PRO®, PRO Boost and DNA do I need to use per 1 liter of culture?
As a starting point, we recommend a starting TransIT-PRO® Reagent:PRO Boost:DNA ratio of 1:0.5:1 (1 ml of TransIT-PRO® reagent, 0.5 ml of PRO Boost Reagent and 1 mg of DNA per mL of culture). Therefore, 1 L of culture would require 1 mL of the TransIT-PRO® Transfection reagent and 0.5 mL of the optional PRO Boost Reagent.

Q10. What is the largest culture volume that you have used for TransIT-PRO® transient transfections?
Through collaboration, we have successfully transfected up to 4 liters of suspension CHO cells in a WAVE bioreactor. Additionally, we have received feedback from customers who have successfully transfected 1 liter bioreactors using the TransIT-PRO® Transfection Kit.

Q11. Will antibiotics interfere with my transfection?
Some antibiotics such as Kanamycin 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).

Q12. Is a media change required post-transfection?
No. A media change is not required following transfection with the TransIT-PRO® Transfection Kit. If desired, a media change can be performed at time points later than 4 hours post-transfection.

TROUBLESHOOTING QUESTIONS AND ANSWERS

Q13. What can I do to further improve transfection efficiency of my target cell type when using the TransIT-PRO® Transfection Kit?
Multiple factors could contribute to low transfection efficiency; 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 0.5‒1.0 × 10⁶ cells/ml at the time of transfection is desired. 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.
Suboptimal PRO Boost to DNA ratio - Determine the optimal PRO Boost:DNA ratio by titrating the reagent from 0 -1.5 µl per 1 µg DNA.
Poor quality of transfecting 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.
Medium formulation not compatible with transfection - Some media do not exhibit good protein yields possibly due to components that inhibit transfection. For details on this topic, please refer to Q14-15.

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

Q14. Can growth media formulation impact transfection performance when using the TransIT-PRO® Transfection Kit?
Yes. Growth medium formulation can have a profound impact on transfection efficiencies. The TransIT-PRO® Transfection Kit displays broad media compatibility but certain media ingredients e.g. heparin, Pluronic F68, etc. may inhibit transfection. If your chosen media exhibits low protein yield, adaptation to compatible media may improve transfection performance.

Q15. Can I add supplements to the transfection media?
Yes. However, we recommend testing your transfection conditions with and without the media supplement to ensure that it does not inhibit transfection. Certain supplements such as 4mM L-glutamine does not impair transfection. If an inhibitory supplement such as Pluronic F68 is needed for long-term growth and increased protein yields, it can be added to the media 18-24 hours post-transfection.

Frequently Asked Questions:

TransIT-PRO® Transfection Kit

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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