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Bioprocessing TUTORIAL Mammalian Transient Transfection System Novel Approach Produces Gram-Per-Liter Expression Levels of Biologically Active Proteins Meredith B. Jones, Ph.D., Chao Yan Liu, Sanjay Vasu, Ph.D., Isabel Cisneros, Henry Chiou, Ph.D., and Jonathan F. Zmuda, Ph.D. Recent advances have allowed transient pro- tein expression to become a fast, flexible, and economical way to produce high-quality recombinant proteins without the time and cost associated with generating stably trans- fected cell lines. The levels of protein generated via tran- sient expression, however, have tended to be significantly lower than those of stably trans- fected cell lines. When large amounts of pro- tein are required, stable cell lines are often still preferred to transient systems. To further increase the utility of transient expression systems, the next key advances will need to approach, or equal, expression levels attained using stable expression sys- tems without losing the speed and flexibility of transient systems. In this article, we report the development of a novel transient transfection system— Expi293™ (Life Technologies; www.lifetech. com)—that utilizes high-density 293F cell cul- tures to generate expression levels greater than 1 g/L for human IgG and non-IgG proteins. System Optimization To obtain multifold increases in protein expression levels over traditional transient systems, it is critical that the transfection step be performed at significantly higher cell densi- ties, allowing for increased volumetric protein Figure 1. Optimization of cell culture medium, cell line, and transfection enhancers: (A) Expi293F Cells were seeded at 0.2 x 106 cells/mL in various culture media and cell density and viability were monitored over time. (B) Human IgG was expressed using the Expi293 Expression System using three different 293F cell lines, (C) with and without the addition of transfection enhancers 1 and 2. A B C production capacity. This requirement of high density cell cultures at the time of transfec- tion necessitated the development of a new cell culture medium able to support extremely high numbers of cells, with high viability, throughout the production run. To this end, a novel chemically defined and animal origin-free cell culture media (Expi293 Expression Medium) was developed that al- lows 293F cells to reach viable cell densities of 14 x106 cells/mL or greater while still main- taining high viability (Figure 1A). In contrast, traditional 293 culture media support a maximum viable cell density of only 4–5 x 106 dia enables transfection at 2.5 x 106 cells/mL. cells/mL. This new cell culture me- cells/mL, compared with traditional protocols that rec- ommend transfection at 1.0 x 106 This increase in the number of viable cells/ mL at the time of transfection enhances the protein production capacity per mL of cul- ture media by 2.5-fold and leads to signifi- cantly higher volumetric protein yields. In addition to allowing for increased vi- able cell densities in culture, cell-specific pro- ductivity was also improved through the gen- eration of a high expressing HEK293F cell line and by the development of transfection enhancers that are added directly to the me- dia post-transfection. To generate the high-producing Expi293F cells, parental FreeStyle 293F cells were adapted into Expi293 ex- pression medium and then selected over time for su- perior protein production under high-density culture conditions. A The resultant Expi293F cells possess an increased growth rate compared to FreeStyle 293F Cells (aver- age doubling times of 21– 24 hours vs. 25–27 hours, respectively), higher viable cell densities, and increased cell viability. The new Expi293F cells produced up to 1.7-times more protein than parental FreeStyle 293F cells used in the Expi293 expression sys- tem (Figure 1B), indicating 50 | October 1, 2012 | genengnews.com | Genetic Engineering & Biotechnology News a significant increase in the specific produc- tivity of the Expi293F cells. Lastly, the transfection reaction itself was optimized through the development of a new transfection reagent (ExpiFectamine™ 293) paired with proprietary transfection enhanc- ers designed to work specifically with this re- agent to further increase overall protein yield by approximately 3-fold (Figure 1C). Gram per Liter Expression When combined into a single expression system and optimized via multi-factorial DoE, the protein yield improvements enabled by each of the Expi293 system components (me- dia, cells, transfection reagent, enhancers), had an additive effect leading to significant increas- es in protein expression levels compared to the FreeStyle 293 expression system. Expression levels of greater than 1g/L were attained for multiple proteins, includ- ing a human IgG and an Fc-tagged Cripto protein (Figures 2A-B). The largest protein yield increase, 11.8-fold, was seen in the ex- pression of a rabbit monoclonal antibody (Figure 2C). A 3.5-fold increase was ob- served for the integral membrane protein `-2-adrenergic receptor (Figure 2D), and a 4.4-fold increase for the secreted growth fac- tor erythropoietin (data not shown). These results were readily scalable, with consistent protein expression levels gener- B C D Figure 2. Comparison of expression levels for four different proteins using the Expi293 system and the FreeStyle 293 System.