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Case Study // SEP 4 2014

High Transient Transfection Levels Translate to Higher Specific Productivity in Suspension CHO cells

A more cost effective solution than linear PEI for transient transfection of CHO-S cells

"Of the three transfection reagents examined and with the efficiency obtained in this study, the cost of the Lipofectamine® 2000 reagent would be the highest at a 1 L bioreactor scale, followed by TransIT-PRO® and then PEI.However, the yield of EGFP protein from TransIT-PRO® would be higher than those from the other two methods.Therefore, in order to produce the same amount of EGFP protein the material cost of using TransIT-PRO® was estimated to be approximately 64% and 87% lower than using 25kDa linear PEI and Lipofectamine® 2000, respectively."

- Sou, et al.

Background

The use of recombinant proteins as biologics has accelerated drug discovery research; complex targets often require post-translational modifications which necessitate the use of mammalian cell hosts.Suspension cells derived from Chinese hamster ovary (CHO) cells are commonly employed due to their desirable growth characteristics, product quality and history of regulatory approval.The ultimate production of the biologic using a stable monoclonal cell population minimizes batch-to-batch variability and reduces cost at an extremely large scale.Generation of stable cell lines is a time and cost intensive process; giving rise to the need for faster methods producing usable amounts of protein for preclinical studies.Transfection methodologies, media formulation, cell line engineering, and vector design are all important factors in enhance yields in upstream development of biologics in suspension CHO cells.

In the present case study, three different methods for transient transfection of CHO-S cells were compared via GFP efficiency, cell viability, gene and mRNA copy number at normal (37°C) and mild hypothermia (32°C) conditions.The authors illustrate how optimized transfection conditions can increase protein yield which translates to cost-effective solutions.

Evaluation of transfection methods for transient gene expression in Chinese hamster ovary cells
1Sou SN, Polizzi KM and Kontoravdi, C. Advances in Bioscience and Biotechnology, 2013 December vol. 4 no. 1013-1019.

What is the role of the transfection reagent in recombinant protein production?

Transfection reagents vary in their ability to condense DNA, interact with the plasma membrane, release of the nucleic acid cargo from the endosome, and nuclear entry.Transfection is a multi-step process that is heavily influenced by variables such as - cell type, plasmid size, growth media and transfection reagent: plasmid DNA ratio.Since the formulations of many commercial transfection reagents is proprietary, empirical testing is required.Sou et al. compared three chemical transfection reagents, polyethylenimine (PEI, Polysciences), Lipofectamine® 2000 (Life Technologies) and TransIT-PRO® (Mirus Bio) by testing a wide range of reagent-to-DNA ratios for each reagent and analyzing the best ratio in more depth.Their investigation utilized a 48 well plate Biolector (m2p-labs) for growth and fluorescence intensity measurements; whereas, cell density and viability were measured by Trypan blue exclusion.

Differences in cell health and viability, DNA and mRNA copy number and GFP intensity were observed between each condition at both temperatures tested. The authors found that Lipofectamine® 2000 had the highest cell viability and growth post-transfection; however, the DNA and mRNA copy number were very low contributing to minimal EGFP expression.GFP fluorescence intensity revealed higher and more sustained expression levels following the transfection with the TransIT-PRO® Transfection Reagent; 7.8 and 2.8 times higher maximum fluorescence compared to Lipofectamine® 2000 and PEI methods, respectively.This was despite the drop in viability in cells transfected with TransIT-PRO® (~65% at day 3) and linear PEI (~60% at day 2) at 37°C implying better cell-specific productivity using the TransIT-PRO® transfection reagent. Differences in EGFP DNA and mRNA copy number were apparent at 37°C but the trends were more obvious at 32°C.

Can mild hypothermia enhance transient protein production yield?

Shifting to a lower temperature post-transfection, typically at 24 hours post-transfection, is frequently used to encourage protein production by maintaining a longer and more viable stationary phase.As stated by the authors, lower temperature is also believed to help stabilize and increase mRNA levels as well as subsequent translation and protein folding.Indeed, Sou et al. found that CHO-S cells transfected with TransIT-PRO® grew moderately and maintained viabilities of 60-70% throughout the 8 day incubation at 32°C; however, cells transfected with linear PEI were unable to recover from the temperature shift to 32°C resulting in 10% cell viability.Mild hypothermia also increased the sustenance of the EGFP DNA and mRNA copy number with the TransIT-PRO® Reagent.Interestingly, linear PEI yielded approximately 1/2 of the DNA copy number (compared to TransIT-PRO®) and very low mRNA levels 3 days post-transfection.Lipofectamine® 2000 had decreased levels of both EGFP DNA and mRNA.GFP expression levels at 32°C achieved higher peak fluorescence and productivity with the TransIT-PRO® reagent compared to both linear PEI and Lipofectamine® 2000.The higher specific productively achieved at 32°C combined with the increased DNA and mRNA copy numbers suggest that the mild hypothermia is effective for protein production in cells transfected with TransIT-PRO®.

This research demonstrates that TransIT-PRO® is an efficient and cost-effective transfection reagent for transient transfection of CHO-S cells.

View more information on large scale protein production.

Samples of the TransIT-PRO® Transfection Kit are available upon request.

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