Rapid, scalable, high-yield protein production from transiently transfected suspension cultures.
- An optimized expression system for CHO and 293 suspension cultures
- Functional proteins with mammalian posttranslational modifications
- Easy scale-up and purification with serum-free and animal origin–free components
The FreeStyle MAX System for CHO or 293 cells
The FreeStyle MAX System is a breakthrough protein production technology for easily scalable, rapid generation of posttranslationally modified, functional proteins in Chinese Hamster Ovary (CHO) or human embryonic kidney (HEK) 293 cells. The cells are adapted to suspension growth in FreeStyle CHO or FreeStyle 293 Expression media, which provide an optimal balance in supporting high cell growth and maximal transfection efficiency. Transient transfection of either cell line is accomplished using the new high-efficiency FreeStyle MAX Reagent.
The choice of either CHO or 293 cells allows greater flexibility to use the most appropriate cell line to produce your recombinant protein of interest. The FreeStyle MAX System for CHO cells reliably produces milligram quantities of human IgG and other proteins (Table 1), and provides an advanced alternative to transient 293 expression systems and conventional stable cell line generation (Figure 1).
The FreeStyle MAX Expression System kits include FreeStyle MAX Reagent, Gibco FreeStyle Expression Media, FreeStyle CHO or 293 cells , and OptiPRO SFM. You can also purchase the system components separately. The media and transfection reagent components of the FreeStyle MAX System are animal origin free and protein free.
Table 1. High protein yield in FreeStyle MAX CHO suspension cells.
| Protein | Day 1 | Day 2 | Day 3 | Day 4 | Day 5 | Day 6 |
|---|
| Human IgG | 6 | 18 | 28 | 36 | 42 | 47 |
| Factor IX | 0.7 | 1.6 | 2.2 | 2.0 | 2.3 | 2.2 |
| HGH | 50 | 109 | 178 | 214 | 225 | 243 |
| EPO | 2.3 | 6.5 | 7.4 | 8.5 | 8.8 | 8.9 |
Human IgG, Factor IX, human growth hormone (HGH), and erythropoietin (EPO) were produced in FreeStyle CHO-S cells. Cells were cultured to a density of 1 x 106 cells/ml in 30 ml of FreeStyle CHO Expression Medium in a 125 ml shaker flask. The transfection complexes were formed using 1.25 μg of plasmid DNA and 1.25 μl of FreeStyle MAX Reagent per ml of culture volume to be transfected, in OptiPRO SFM. At 24 hours post-transfection, transfection efficiency was 60% as measured by GFP expression. Protein production was measured by ELISA, in mg/L.
The FreeStyle 293 System for 293 cells
The FreeStyle 293 System for easily scalable, rapid protein production generates functional, posttranslationally modified, proteins in human HEK293 cells. The system offers exceptional productivity and speed in the transient expression of recombinant antibody proteins (Table 2). Transfection is performed using the high-efficiency 293fectin transfection reagent. The transfection protocol can be readily scaled with minimal or no modifications for suspension culture volumes from less than 30 milliliters to greater than 10 liters while maintaining equivalent volumetric protein yields (Figure 3).
The FreeStyle 293 System kit includes 293fectin transfection reagent, Gibco FreeStyle 293 Expression Media, FreeStyle 293 cells, and Opti-MEM I medium. You can also purchase the system components separately.
Table 2. High protein yield in FreeStyle 293 suspension cells using 293fectin reagent.
| Protein | Day 1 | Day 2 | Day 3 | Day 4 | Day 5 | Day 6 |
|---|
| Human IgG | 12 | 63 | 99 | 128 | 159 | 164 |
| Factor IX | 0.34 | 0.73 | 0.92 | 0.98 | 1.0 | 1.1 |
| HGH | 19 | 150 | 67 | 143 | 163 | 181 |
| EPO | 0.03 | 0.05 | 0.08 | 0.1 | 0.11 | 0.14 |
Human IgG, Factor IX, human growth hormone (HGH), and erythropoietin (EPO) were produced in FreeStyle 293-F cells. Cells were cultured to a density of 1 x 106 cells/ml in 30 ml of FreeStyle 293 Expression Medium in a 125 ml shaker flask. The transfection complexes were formed using 1.0 μg of plasmid DNA and 2.0 μl of 293fectin reagent per ml of culture volume to be transfected, in OptiPRO SFM or Opti-MEM I medium. At 24 hours post-transfection, transfection efficiency was >75% as measured by GFP expression. Protein production was measured by ELISA, in mg/L.
Convenience and speed
Large-scale, transient transfection of mammalian cells in suspension culture is a reliable and rapid method to generate milligram quantities of mammalian recombinant proteins for functional, therapeutic, and structural studies. With the FreeStyle MAX and FreeStyle 293 System, you only need a sterile work bench, a CO2 incubator, and a platform shaker with shake flasks or a spinner culture. Stable cell line development requires labor-intensive screening and adaptation procedures to identify highly expressing clones.
| | 
| | The overall process requires months from the initial selection of a stably transfected cell line to production of final product. In comparison, the FreeStyle System achieve large-scale protein production in days, and is easily scalable from 30 ml to 10 L cultures (Figure 2 & 3). Detailed protocols for scale-up are available at here. |
Protein production services
FreeStyle Custom Services are also available to produce your proteins under optimized conditions in suspension CHO or 293 systems based on the scale you request. We offer time-course studies, and protein production in 1–50 L cultures. We can provide you with the cell pellet, or even better, the purified protein, as the final deliverable. Contact your Invitrogen Sales Representative to discuss your needs and to initiate a project.
Journal Publication from HighWire Press
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