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 densities, allowing for increased volumetric protein production capacity. This requirement of high density cell cultures at the time of transfection 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 allows 293F cells to reach viable cell densities of 14 x106 cells/mL or greater while still maintaining high viability (Figure 1A).
In contrast, traditional 293 culture media support a maximum viable cell density of only 4–5 x 106 cells/mL. This new cell culture media enables transfection at 2.5 x 106 cells/mL, compared with traditional protocols that recommend transfection at 1.0 x 106 cells/mL.
This increase in the number of viable cells/mL at the time of transfection enhances the protein production capacity per mL of culture media by 2.5-fold and leads to significantly higher volumetric protein yields.
In addition to allowing for increased viable cell densities in culture, cell-specific productivity was also improved through the generation of a high expressing HEK293F cell line and by the development of transfection enhancers that are added directly to the media post-transfection.
To generate the high-producing Expi293F cells, parental FreeStyle 293F cells were adapted into Expi293 expression medium and then selected over time for superior protein production under high-density culture conditions.
The resultant Expi293F cells possess an increased growth rate compared to FreeStyle 293F Cells (average 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 system (Figure 1B), indicating a significant increase in the specific productivity 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 enhancers designed to work specifically with this reagent to further increase overall protein yield by approximately 3-fold (Figure 1C).