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.
In an experiment where FreeStyle CHO-S cells were transfected with the GFP reporter plasmid at a range of starting cell densities from 3x105 per mL to 1x106 per mL, the percentage of GFP-positive cells one day post-transfection, decreased as the starting cell density increased (Figure 1A). When the total number of GFP-positive cells was determined at various time points up to seven days post-transfection, however, the greatest total number of GFP positive cells in the culture was achieved when the highest starting cell density was used (Figure 1B).
When IgG yields were examined using the same range of starting cell densities, it was found that the highest yields resulted from the highest starting cell density (Figure 1C). These findings indicate that a critical factor for successful high-level r-protein production is the ability of the transfection reagent and medium combination to support transient transfection and sustained cell growth at relatively high cell densities.
Over the time course of IgG production, the cell density in a shake flask containing a 30 mL culture volume increased from 1x106 per mL to almost 4x106 per mL. Cell viability was above 90% at 24 hours post-transfection and was still above 85% at six days post-transfection, without medium change or supplementation (Figure 1D).
Similar IgG yields were obtained when shake flasks containing 1 L of culture medium and a total starting culture of 1x109 cells were used. This observation demonstrates the ease with which protein production can be scaled up using the FreeStyle-MAX CHO System.
Prior to scaling up transfection experiments, expansion of cell cultures is required. While cell growth and transfectability of cultures was maintained for as many 40 passages (more than three months), it has been observed that at later passages, protein yields may be reduced. Therefore it is recommended that cultures are not maintained for more than 30 passages prior to transfection.
Erythropoietin and Factor IX
In addition to IgG, production of two other therapeutically relevant proteins was examined: human erythropoietin (EPO), a heavily glycosylated protein used to increase red blood cell mass, and coagulation Factor IX, a glycoprotein used in the treatment of hemophilia B. Each gene was cloned in the expression vector pcDNA3.3-TOPO® then transfected into FreeStyle CHO-S cells. Transfection and cell culture conditions previously outlined were used.
Over a time course of eight days post-transfection, yields of over 35 mg/L of EPO and 8 mg/L of Factor IX were obtained (Figure 2), which were higher than the yields obtained using HEK293 cells. Transfection efficiency measured using a GFP reporter, though, was similar. These differences may be the effect of factors such as variations in the average number plasmid molecule delivered per cell for the two cell types as well as differences in the levels of transcription or translation. In addition, variations in the efficiency of post-translational modification and secretion of the two proteins may play a role.
While much of the FreeStyle-MAX CHO System development was done using small-scale shake-flask cultures, many of the technical parameters identified in these studies are relevant to larger-scale transfections. The kinetics of DNA-transfection reagent complexation appear similar, with the same DNA and transfection reagent concentrations and complexation times being optimal for both a 30 mL shake-flask culture and a 10 L culture in a Wave Bioreactor disposable cell culture system. These studies are ongoing and are likely to result in further enhancements to the value of the FreeStyle-MAX CHO System for protein production in larger-scale cultures.