October 15, 2015 (Vol. 35, No. 18)

Novel Transient Gene Expression Approach for Increasing Transfection Efficiency

Transient gene expression (TGE) is commonly used for medium-scale production of recombinant proteins and antibodies. This approach allows generation of sufficient protein amounts, thus avoiding a major investment in production of stable cell lines prior to proof- of-concept studies or tools validation.

Indeed, the speed and flexibility of TGE has enabled this technique to be widely accepted for early discovery, research applications, and process development. However, the protein yields usually remain lower with TGE than with stable gene expression processes. In addition, CHO cells, which are widely used in bioproduction, are especially difficult to transiently transfect with chemical reagents.

One of the TGE manufacturing strategies for improving productivity is to increase transfection efficiency. Polyplus Transfection has developed a novel transfection solution, FectoPRO®, which allows generation of superior protein amounts and viral titers.

To develop FectoPRO, screening of many chemical structures was performed in which physicochemical and biological properties were evaluated. Suspension CHO cells were transfected with a reporter plasmid system (GFP-Luc plasmid) to determine the cell viability, transfection efficiency, and protein productivity. (Figure 1, left panel). A comparison of FectoPRO candidates to some current commercially available reagents was performed according to protocol recommendations (Figure 1, right panel).

The best hits were then screened for the ability to provide the highest protein production yield with the lowest DNA amounts used. After selection of the best molecule, the protocol was further optimized by choosing the most appropriate DNA amount and, additionally, a booster was developed to ensure unmatched production yields with low amount of DNA transfected in both CHO and HEK293 cells.

All of the optimizations performed led to the design of a novel solution for improved protein yields in TGE systems.

Figure 1. Methodology of screening . To select for the best candidates, CHO cells were seeded at 1 × 106 cells/mL in 30 mL of medium and transfected using pCMV-EGFP-Luc plasmid (containing both GFP and Luciferase). We assessed cell viability by measuring the percent of trypan blue cells, transfection efficiency was evaluated by quantifying the percent of GFP positive cells and protein yields were determined by measuring luciferase production 24 h post-transfection. Transfections were performed using several FectoPRO candidates and FectoPRO Booster (0.5 µg DNA/mL), Reagent A (1.25 µg DNA/mL), or Reagent B (1 µg DNA/mL) following recommended protocols for the respective reagents.

Current Challenges in Bioproduction

Regardless of the cells and medium used or the final protein/antibody produced, the majority of biomanufacturers want to produce proteins with good glycosylation profiles and reach high protein yields. While working on this goal, they should ideally be able to maintain flexibility and scalability in their processes and ultimately meet the demands with reduced costs.

Challenge 1. Reaching Good Transfection Efficiency in Suspension Host Cells (CHO and HEK293)
One of the initial goals when performing transient gene expression is to achieve good transfection efficiency. A high percentage of transfected cells is a prerequisite for obtaining good protein yields later. Cells that are commonly used for production of large quantities of protein or antibody are usually grown in suspension and are usually more difficult to transfect than adherent cells. This is especially the case with suspension CHO cells that are predominantly used if the goal is to obtain a protein with a good glycosylation profile.

Improving transfection efficiency of suspension CHO cells and increasing protein yields of highly glycosylated proteins represents one of the main challenges in bioproduction.

We compared FectoPRO to some other commercially available transfection reagents by transfecting suspension CHO and HEK293 cells with GFP plasmid. The transfection efficiency obtained was quantified by analyzing GFP positive cells 24 h after transfection. In combination with its proprietary booster, FectoPRO reagent shows higher transfection potency and leads to significantly improved transfection efficiency in both CHO and HEK293 cells (Figure 2). This improvement allows more extensive use of CHO cells in protein production and in early-stage development.

Challenge 2. Improving Protein Yields in TGE
Protein yields are directly influenced by transfection efficiency. Thus, improved transfection efficiency not only in CHO but also in HEK293 cells leads to enhanced production yields in these cells.

Figure 2. Transfection efficiency obtained with several reagents in suspension CHO and HEK293 cells. Suspension CHO and HEK293 cells were seeded at 1 × 106 cells/mL in 30 mL of their recommended media and transfected using a GFP-expressing plasmid with FectoPRO® ± FectoPRO® Booster (0.5 µg DNA/mL), Reagent A (1.25 µg DNA/mL), or Reagent B (1 µg DNA/mL) following recommended protocols for the respective reagents. GFP expression was evaluated 24 h after transfection using fluorescence microscopy.

We first assessed productivity in suspension CHO cells by producing an Fc-fragment of IgG3 antibody. Results were analyzed either by HPLC for quantitative analysis (Figure 3A) or Western blot for qualitative analysis (Figure 3B). FectoPRO largely outperforms other transfection reagents commonly used in bioproduction processes in terms of protein yields.

After that, we wanted to assess the protein yields when full antibodies were produced over longer periods of time in suspension CHO cells (Figure 3C). In this case, suspension CHO cells were transfected in order to produce complete mouse antibody and the two conditions tested with FectoPRO (0.5 µg of DNA/mL and 0.8 µg of DNA/mL) gave excellent results.

Challenge 3. Scalability, Flexibility, and Ease of Use
Performing transient transfection and producing proteins at various scales with one reagent by using the same conditions is highly desirable as it allows users to save time usually spent on optimization and focus on achieving the desired goals.

The FectoPRO-mediated transfection process is easily scalable from a few milliliters to several liters of cell culture, ensuring reproducible protein production (Figure 3D). FectoPRO simplifies the bioproduction process with a protocol that can be easily adapted to different culture vessels and thus allows the use of one reagent from early-stage development up to therapeutic protein production.

Another important parameter that allows flexibility when producing proteins in TGE is freedom to choose the desired serum-free medium. FectoPRO works efficiently in a wide range of commercially available serum-free media with minor protocol adjustments.

Challenge 4. Reducing Costs and Meeting the Quality Requirements
High-quality plasmid DNA preparation can be costly, especially when large DNA amounts are required for large-scale protein production. FectoPRO saves on DNA cost by using only 0.4 to 0.8 µg DNA/106 cells/ mL of cell culture.

FectoPRO is chemically defined and guaranteed free of animal-origin components. Systematic lot management and release testing is performed for each lot produced. The FectoPRO transfection kit, which undergoes advanced quality controls for protein productivity, cell viability, and complete sterility, is in compliance with biomanufacturing guidelines.    

Figure 3. Improved and scalable production of Fc fragment or full IgG antibody obtained with several reagents in suspension CHO cells. CHO cells were transfected with IgG3-Fc expressing plasmid using reagents and conditions mentioned. Antibody quantification was performed by using protein G affinity column (HPLC) (A) and qualitative analysis was done by Western blot 24, 48, and 72 h after transfection (B). To assess the production yields of a full antibody over longer period of time, CHO cells were co-transfected with plasmids coding for the heavy and the light chains of mouse IgG with FectoPRO reagent or Reagent A using conditions mentioned. Quantification was performed by using protein G Biosensors (FortéBIO) at several time points (24–120 h) after transfection (C). To compare production yields in different volumes of cell culture suspension CHO cells were seeded at 1 × 106 cells/mL in either 30 mL, 100 mL, or 1 L of the media and transfected with IgG3-Fc expressing plasmid using FectoPRO + FectoPRO Booster (0.5 µg DNA/mL). Quantification was performed 1, 2, and 3 days after transfection using protein G affinity column (HPLC) (D). [Data for Figure 3C were kindly provided by ProteoGenix.]

Mathieu Porte is project leader, bioproduction, Valerie Kedinger, Ph.D., is a scientific and technical support specialist, Patrick Erbacher, Ph.D., is CSO, and Jelena Vjetrovic, Ph.D. ([email protected]), is a scientific and technical support specialist at Polyplus Transfection.

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