Optimizing mammalian cell culture for volumetric productivity involves working on different levels at different stages of the product’s lifecycle: cell line genetic engineering, clone selection, nutrition (media, supplements, feed), and the process itself are all reasonable places to intervene. The first two activities fall under the category of nature since they circumscribe innate capabilities, whereas the latter involve nurture.
Ten years ago one might have described optimization efforts as holistic since the goal of cell optimization is overall improvement, not necessarily achieving the highest specific productivity per cell (through nature) or cell density (nurture). Developers often find that they must play one factor against others to achieve the right mix. “For constitutive expression systems, devoting resources toward expression may divert them from cell growth, which can result in overall lower productivity,” says James Blackwell, Ph.D., senior consultant with Bioprocess Technology Consultants. Similarly, lowering the temperature of a cell culture from optimal may slow cell division but improve volumetric productivity by directing cells toward production rather than reproduction.
Thomas Seewoester, Ph.D., director of process development at Amgen, notes that cell culture process yields are defined by total viable cell mass and cell-specific productivity. “To optimize a process, both factors need to be maximized and sometimes balanced against each other,” he says.
Large companies like Amgen possess considerable in-house optimization skills to cover cell engineering, clone selection, media and feed compositions, and process parameters. Its experience with a large number of similar molecules has enabled Amgen to establish process platforms that avoid extensive optimization at each level and each molecule early in process development. Later on, each product receives targeted media- and process-level optimization to achieve productive, cost-effective, robust processes. Still, the company works with external collaborators and partners to explore new technologies and novel approaches to cell process optimization.
Cells vs. Microorganisms
Nurture tends to provide greater benefits compared with nature for mammalian cells, while the opposite is true for microbial cultures. Thus, for mammalian cells, increasing biomass is the key to improving volumetric productivity, while for yeast the answer is genetic engineering. “It is possible to engineer characteristics into yeast in ways that are not possible with mammalian cells,” notes David Robinson, Ph.D., vp for bioprocess R&D at Merck Research Laboratories.
Yeast possess much simpler genomes than do mammalian cells—approximately 6,000 genes versus 20,000. “Plus, the number of tools to do genetics in yeast is higher,” Dr. Robinson adds. Merck owns the rights to yeast-based protein expression technology based on Pichia pastoris through its acquisition of GlycoFi in 2006.
Merck is excited about Pichia because this expression system allows developers to control glycosylation patterns in proteins where this is significant. Glycosylation and other post-translational modifications are among the crucial issues facing developers of biosimilars. “Control over glycosylation provides us with the ability to do product engineering as opposed to simply productivity enhancements,” says Dr. Robinson, who uses the term “bio-better” to describe proteins whose glycosylation patterns provide superior properties such as circulating half-life, tissue targeting, and biological activity. “It has the potential for therapeutic protein development that has never before existed.”
Merck also employs Lonza Biologics’ glutamine synthetase technology, GS System™, which uses a viral promoter and selection via glutamine metabolism to provide stable, high-yielding mammalian cell lines. GS System is used by more than 85 biotechnology companies. Five approved products, including Zenapax® (Roche) and Synagis® (MedImmune), use the technology, which allows developers to reach the 50 pg/cell/day production level, according to Lonza Biologics. From there, Merck scientists increase biomass through standard fed-batch techniques.
According to Dr. Robinson, 50 pg/cell/day is a kind of soft practical upper limit for cultured monoclonal antibody-producing cells. Some produce more, but those tend to be outliers. Dr. Blackwell notes that secreted antibody levels from B cells in response to a pathogen challenge, on the order of 100 pg/cell/day, may suggest a true biological ceiling for antibody production in mammalian cells.