Josh P. Roberts has an M.A. in the history and philosophy of science, and he also went through the Ph.D. program in molecular, cellular, developmental biology, and genetics at the University of Minnesota, with dissertation research in ocular immunology.
Scaling up protein production involves more than just doubling the number of T-flasks in your incubator. Whether you just want to take your basic research up a notch or are ready to do animal studies or a small Phase I trial, chances are you’ll need to take your protein production a little more seriously, as well.
As the projects get bigger, you’ll probably want to get the most out of the time and investment that goes along with scale-up. Figuring out the most efficient ways to do that—from engineering the cell line and culture system to harvesting and purifying the final product—can be well worth the effort. Here we explore ways to say goodbye to “good enough” and hello to optimized protein production.
Adherent cells: Sticky business
“Everything in research is going smaller, smaller, smaller,” says research associate Fred Schendel, who leads the large-scale protein-purification efforts at the University of Minnesota Biotechnology Resource Center (BRC). “Techniques have gotten down where you don’t need much protein to do [basic] research anymore.” Citing high costs and decreasing demand, Schendel says the BRC has eliminated its mammalian cell-culture equipment.
But for those who still need hundreds of milligrams of protein, there are ways to increase your lab’s production. Or, adds Schendel, you can outsource. “There are a lot of contract labs that would make significant amounts of material for you.”
So how do you scale up? Many labs are used to culturing adherent cells in multiwell plates, Petri dishes and T-flasks. It’s relatively straightforward to use larger flasks or to squeeze a bit more production from your incubator’s real estate with multilayered cell-culture flasks (such as Thermo Scientific’s Nunc™ TripleFlasks™, EMD Millipore’s Millicell® HY multilayer flasks and Corning’s 10-layered HYPERFlask) or connected, stackable trays (such as Thermo Scientific's Nunc Cell Factory™ and Corning’s CELLStack systems).
At times, demands for protein may outstrip a lab’s capacity to grow enough adherent cells. One alternative is to buy more incubators or scale up to roller-bottle or other systems that require capital investment—and personnel to deal with them.
At the Sanford-Burnham Medical Research Institute, “most of the time we try to put everything in suspension cultures, because it scales up more easily,” says protein production and analysis core-facility manager Darrin Kuystermans. That typically means engineering a suspension-adapted cell line to produce the protein of interest, if possible. As a last resort they will use microcarrier beads—available from a variety of sources including Corning Life Sciences and GE Healthcare Life Sciences—to grow the adherent cells as a suspension culture.
In or out?
Another way to boost protein production is to have the cells secrete the protein into the culture medium, rather than retaining it, as that makes downstream processing far less arduous. “We can actually clarify it from the cell medium, so we don’t have to burst open the cell and deal with all the other proteins in there during the protein-purification part,” explains Kuystermans.
Occasionally a protein won’t secrete, despite having a secretion signal. In such cases, Kuystermans may engineer in a purification tag that enables him to fish the protein out of a messy cell lysate more easily.
Kuystermans recommends testing the protein-expression culture in a small shake-flask setup, rather than going directly into a larger-scale system, “until you know if the cells are adapted to that kind of condition with the protein being produced.” Agitation introduces extra oxygen, induces shear and can create other stresses that may cause the cells to start clumping or otherwise prevent them from growing in even larger suspension cultures. For cells or proteins you have doubts about, it might even make sense to start out with a transient transfection rather than spending months on a stable expresser and then find out the system is not working.
Optimize
After you know the expression system is working, it’s important to optimize conditions before embarking on a pilot-scale project. The most important culture parameters are temperature, dissolved oxygen (DO), pH and glucose—these can be monitored using probes and sensors, and controlled for either automatically or manually, depending on the system. One way to optimize these is in what’s called a “micro-bioreactor” or “scale-down model”—either a commercial system, such as Applikon Biotechnology’s micro-Matrix or m2p-labs’ BioLector—or simply in an array of shaker flasks. Here various conditions, including feeding, timing and atmosphere, can be compared.
Of course, not everything scales linearly, says Mark Hirschel, former chief science officer of BioVest International, which offers custom biomanufacturing and purification services. For example, development work typically is performed in batch mode; however, production could be conducted in a higher-density perfusion mode, using hollow fiber reactors, in which case “some of the characteristics of your culture are different, meaning you might have a different outcome.”
Similarly, liquid surface-to-volume ratio often doesn’t scale directly—affecting DO and CO2 (and therefore pH) levels—nor do agitation and aeration.
It’s equally important to optimize the downstream (harvesting and purification) process, beginning with screening (generally serum-free) media that will enable the best separations [1]. Other considerations include stability of the media under separation conditions, rigidity of the resin and column dimensions. For instance, to ensure a constant residence time of the target protein on the column during scale-up, the column diameter can be increased, but the bed height should remain constant, says Kuystermans.
Shake, rock and roll
Scaling up suspension cultures can be done in reusable bioreactors that Kuystermans calls “clean and play” systems—mainly stirred flasks or tanks with the ability to monitor and control various parameters.
But what may be more common these days, especially in an industrial or commercial setting, is to use disposable bag bioreactors, such as the WAVE system from GE Healthcare Life Sciences, or similar products from vendors such as Sartorius, Pall and others, notes Hirschel. “There are several systems out there that work on a small scale,” he says. These include liners for stirred tanks as well as disposable spin flasks in a range of sizes.
Wilson Wolf Manufacturing has developed the static G-Rex®, which is essentially a T-flask with a silicone membrane on the bottom for passive gas transfer. This lets cells grow at relatively high density, explains Hirschel, who is now chief operating officer of Wilson Wolf. “By adding a lot of media to that, letting it sit for a week, you get a significant antibody production”—in the range of 500 to 600 mg per 500 to 1,000 ml of volume, which is approaching the scale of a 20-L bioreactor. “It’s just another approach in the repertoire—a significant step up from T-flasks and extremely appropriate for the university researcher to scale up.”
Protein production is always tricky, and scaling it adds to the complication. It takes some optimization to get everything right. But after you nail everything down, and you have more protein than you know what to do with, you’ll be glad you did.
Reference
[1] Milne, JJ, “Scale-up of protein purification: Downstream processing issues,” Methods Mol Biol, 681:73–85, 2011. [PubMed ID: 20978961]
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