Using mammalian cells for recombinant protein (r-protein) production is often required to obtain the necessary solubility, folding, and post-translational processing for a variety of applications including preclinical, biochemical, biophysical, and drug discovery studies. This approach, however, can be laborious, expensive, and time consuming.
While transient transfection of mammalian cells grown in monolayers can generate significant amounts of r-protein in a short period of time, the scalability of this process is limited by culture surface availability. As a result, large-scale transient transfection of mammalian cells grown in suspension culture has become of great interest.
The most commonly used cell types, human embryonic kidney (HEK) 293 cells and Chinese hamster ovary (CHO) cells, are easily adapted to suspension culture. Protein yields reported for CHO cells have typically been lower than those seen with 293 cells, likely due in part to the incompatibility of the transfection reagents used and components of CHO cell culture medias. CHO cells, however, are currently the predominant mammalian cell type for protein manufacturing.
Invitrogen (www.invitrogen.com) developed the FreeStyle™-MAX CHO Expression System to allow better alignment between upstream preclinical studies and downstream protein manufacturing.
This system comprises a CHO cell line adapted for suspension culture as well as an animal origin-free medium and transfection reagent. It is scalable and has been tested using shake-flask cultures of up to 1 L volume and fixed-tank as well as disposable 10 L Wave Bioreactors®.
One of the main advantages of the FreeStyle-MAX CHO Expression System is that cell manipulations or growth media changes post-transfection are not required. In fact the same general transfection procedure can be followed irrespective of the final culture volume.
Plasmid DNA is diluted in Opti-Pro™ SFM medium using 1.25 mg of DNA per mL of final culture volume, and the FreeStyle-MAX transfection reagent is also diluted in Opti-Pro SFM medium, using 1.25 mL of transfection reagent per mL of final culture volume. The diluted DNA and transfection reagent are combined, incubated for 20 minutes, and then added drop-wise to the flask. Protein production is then tested, typically over a time course of one to six days post-transfection.
Evaluating Protein Production
The FreeStyle-MAX CHO System was first evaluated using a GFP reporter plasmid to determine the percentage of transfected cells in a population. Various parameters were optimized, including cell density at the time of transfection, DNA and transfection reagent concentrations, as well as DNA-transfection reagent complexation time.
Protein production using human IgG heavy and light chain genes were then assessed. These studies revealed that optimal IgG production did not simply correlate with maximized transfection efficiency, as indicated by the percentage of GFP positive cells.