Optimizing Protein Yields
UCOEs were initially isolated and evaluated by incorporation into vectors for the expression of enhanced green fluorescent protein (EGFP) from the hCMV promoter (Figure 1). Evaluations of the vector were initially conducted in CHO-K1 and after selection and isolation, each clone was analyzed for expression by flow cytometry.
Figure 2 illustrates the high percentage of cells that produce detectable levels of EGFP when compared to clones containing the same vector without the UCOE. It is clear that on an individual basis, cells containing the UCOE vector demonstrate higher production levels than cells containing the vector without a UCOE. This dramatically reduces the number of clones that therefore need to be screened.
The stability of expression from a UCOE vector has been demonstrated by studying the expression of an IgG1 during continuous cell culture of a suspension CHO-S cell line over 50 generations, with expression yields being determined at the start, middle, and end (Figure 3). It can be clearly seen that expression is stable for these higher expressing clones.
UCOE technology enabled the selection of high expressing clones capable of producing 1.5–2 g/L of protein within ten weeks. This compares favorably to traditional methods that can take up to 10–12 months. Figure 4 shows that productivities can be increased from around 0.4 g/L to 1.5–1.9 g/L with minimal optimization in protein-free media with supplemental feeds.
The technology therefore enables not only the isolation of high expressing clones but can also allow the selection of high expressing pools, produced by transfecting the culture with the UCOE expression plasmid, followed by antibiotic selection. Such pools can be isolated within 2–3 weeks of transfection with a minimal quantity of plasmid DNA.
These pools can be used to generate research cell banks without the need for repeat transfection, and demonstrate high levels of productivity up to orders of magnitude above that achievable with transient transfection. This has allowed Cobra to supply large quantities of material for early-stage downstream purification (DSP) development for a number of clients.
In one example a human IgG1 was expressed in an initial shake flask media screen to 645 mg/L; this was followed by a 5 L fermentation, which allowed the development of a purification scheme prior to the isolation and generation of material from clones.
An IgG1 fusion has also been expressed at 500 mg/L in shake flask cultures using a generic medium developed for our CHO-S cell line; this has again allowed the development of an early-stage DSP process with the supply of sufficient quantities of material for activity studies. The production of material in pools is now routinely used at Cobra to reduce project timelines significantly.
UCOE technology is of huge value to the bioprocess industry if combined with informed cell-line development.
The engineering of expression vectors containing UCOEs enables the rapid and easy production of stable, high-expressing cell lines that are suitable for large-scale production and the rapid isolation of high expressing pools. Protein yields of 2 g/L can now be obtained with a reduced drain on resource, providing a cost-effective production solution to the biopharmaceutical sector.