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Creating a kinder, gentler manufacturing process that doesn't kill the product is the goal of process developers doing large-scale cell culture for cell therapy.
This story is about a technology so new, it hasn't been invented yet. Not quite. It's so new that "Googling" the search term "cell manufacturing" pulls up largely irrelevant entries—ones that pertain more to computer hardware configuration than to the large-scale culturing of cells. And yet it is based on traditional cell culture, one of the oldest technologies in biology and the foundation on which so many modern biological discoveries rest.
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Although small-scale cell culture has been around for about a century, the notion of culturing cells on a large scale is only a decade old, invented out of the necessity to accommodate the growing commercial success of biotech-based therapeutics, mostly monoclonal antibodies and small peptide-sized molecules.
Protein drugs can theoretically be synthesized in a laboratory, but the procedure would be painstaking, labor intensive, and expensive. It is far more efficient to follow nature's lead and produce proteins inside the cells.
Figure 1: CompacT SelecT is a medium-throughput automated cell-culture system made by The Automation Partnership.
The industrial route for making protein drugs inside cells is fairly well established, even at the relatively callow age of ten. In the general paradigm, a gene for a therapeutic protein is inserted into a cell—the most popular mammalian cell for industrial purposes being a line of Chinese hamster ovary (CHO) cells. Great quantities—thousands of liters—of the genetically engineered CHO cells are cultured, fed, and nurtured and encouraged to undergo successive cell divisions to make yet more cells, which, of course, are also producing large quantities of the therapeutic protein. The therapeutic protein is secreted from the cells, or the cells are later cracked open so the therapeutic protein can be extracted and purified from them, formulated, packaged, and then sold as a drug.
Either way, the cells are disposable, used only to produce protein product before they're discarded. The techniques used to culture them are rather harsh, by cell-culture standards. "You beat the heck out of [the cells] to get the product out," says Chris Mason, an academic researcher at the Regenerative Medicine Bioprocessing Unit at University College in London. "You can treat the cells as badly as you like," he says, "just as long as they produce the bioproduct."
Figure 2: An automated cell-culture system includes a media pump and circulation pump to push fresh medium and oxygen through the CellCube module and to eliminate spent medium. Ideally, cells within the module are exposed to uniform amounts of gases and nutrients.
Treating the cells badly means that rather than allowing them to rest on a solid substrate, as most cells "prefer," cells grown for bioprocessing are suspended in a kind of nutritive broth called medium. The suspension is contained inside either a large stainless-steel vat or a large plastic (and therefore disposable) bioreactor and somehow rocked or churned or mixed to make sure that all cells have equal access to the nutrients and gases dissolved within the medium. Because they're suspended in huge vats or bags, and because they're being roiled around, the cells ball up and bump up against each other and against the vessel in which they're growing. And, as Mason noted, this harsh treatment—destructive to many cells—is fine, because the cells are not the end game. They're not the product.
At some point, cells will be the product. Regenerative medicine, the ability to repopulate unhealthy organs and tissues with healthy cells, is getting closer to the clinic. Mason detects a major change in interest among venture capital groups, who, he says, "now see some real commercial pressure." For their part, cell-therapy companies are also focusing more on commercializing the technology as opposed to previous years, when they were mostly researching it. Several biotech companies are either preparing to enter or are already in clinical trials with cell-based therapies. Geron (Menlo Park, CA), a biotechnology company focusing on regenerative medicine, is preparing to enter its first clinical trials with a GRNOPC1, a glial-cell product that promotes spinal-chord repair. And other cell-therapies in the service of regenerative medicine are not far behind.
In preparation for the day cell therapy comes into commercial fruition, process engineers, cell biologists, and equipment manufacturers are working out the details of large-scale cell culturing to produce cells as end products. One thing they do know, says Mason, is that for regenerative medicine, "the process is the product. You have to take care of the cell all the way through the process." In other words, culturing cells on a large scale requires a kinder, gentler process than does culturing them to produce protein products. And there's the rub.
By it's very nature, industrial-scale manufacturing requires batch handling, bulk movement, and bulk treatment, which is not necessarily known for its gentleness. And cells, especially the primary and stem cells used in cell therapy, are fragile and require kid-glove treatment.
Until recently, that kind of treatment could be maintained only when a technician performed all of the harvesting, seeding, feeding, waste removal, and packaging manually. And that was fine, even for cell therapy. In its initial iteration, cell therapy involved a patient's donation to himself of his own cells—a process called autologous cell transplant. Healthy white blood cells, for example, are often removed from patients undergoing high-dose anticancer chemotherapy, which kills all remaining white blood cells in the bone marrow—including the cancer cells. While the healthy cells remain outside the body, they are cultured to increase their numbers to a point where they can repopulate the patient's bone marrow once the course of chemotherapy has been completed. Other organs may also be regenerated by similar autologous transplants. Autodonation of cells sidesteps the problem of rejection that comes in transplanting cells from one person into another.
Viewed as a manufacturing problem, however, the batch size of an autologous cell dose is very small. Which does not mean the process cannot be automated. If transplants are being prepared for just one patient, there's little motivation to increase production scales. But imagine a transplant facility, in which many different cell types harvested from many different patients are being cultured for reimplantation at the same time. In that case, the challenge for process engineers is the parallel growth and cultivation of many different cell lines in a sterile, automated setting without contaminating one cell line with the next. Economies of scale can be realized in this setting by multiplexing the cultivation of cell lines.
The real opportunity to develop a large-scale process comes as researchers consider the possibility of allogeneic transplants. Here the idea is that it may be possible to create some kind of "universal" cell that can safely be transplanted in many recipients without concern for rejection. In some scenarios, the cells may be modified as to not express markers that stimulate rejection by the immune system. In others, cells may be typed in a system similar to blood typing, which allows certain classes of cells to be transplanted from one group to another without the requirement of an exact tissue match. However it's achieved, the allogeneic model will require large batches of cells to be cultured for use in cell therapy. And here's where the issues become interesting.
As researchers work to invent an industrial process to "manufacture" cells on a large scale, they draw their experience and knowledge—as well as their assumptions—from two sources. The first is the manual process by which cells have been cultured for a century. And the second comes from their experience with the bulk processing of cells for bioprocessing. What is emerging is a process that Mason likes to call "regenerative medicine 1.0," or "RegenMed 1.0," for short.
From manual cell culture, investigators know that the vast majority of cells grow best when they are attached to a solid surface. They also know that the health of the cells depends on having the proper nutrients in the culture medium, proper gas exchange, and the proper density. If too many cells crowd the plate, some may be induced to stop dividing and enter a differentiation stage where they eventually take on the characteristics of the mature cell type. This would be enormously counterproductive, because cell therapy almost always involves some kind of immature cell type to allow the recipient tissue to continue to regenerate itself. Cell culture also requires someone to remove spent medium, which is devoid of nutrients and replete with the waste products of cellular metabolism. It requires someone to recognize whether cells are viable and when the density dictates that cultures be divided onto new culture plates—a process akin to repotting plants to give them more growing room. Of course, someone also has to remove the cells in the end, package them, transport them, and then implant them in the patient. All of these processes have to be automated.
From bioprocessing, scientists already have gained a lot of general knowledge. They know, for example, how to work in a closed system; they know quality assessment and validation techniques. "There exists a framework in which to do process development that wasn't there when bioprocessing was developed," says Jon Rowley, director for cell therapy research and development at Lonza (Walkersville, MD). "A lot of the equipment has been developed, as well," says Rowley. "Sterile connect technology, for example. We don't have to develop these technologies now from the ground up."
The immediate challenge as Rowley sees it is that "many cells in the cell-therapy world are adherent and don't grow in traditional [for bioprocessing purposes] suspension systems, which means that that part of the [bio]process is not transferable." The next challenge is the need to build an adhesion system that limits batch size. A state-of-the-art vessel for bioprocessing can hold as much as 20,000 L of cell suspension. "There is no 20,000 L culture dish for adherent cells," he says. And then he notes that with current technology, the highest adherent batch size is 6 L, "a far cry from 20,000," Rowley adds.
Process developers have been working closely with equipment manufacturers to overcome the volume problem. In the manual paradigm, cells adhere to the sides of flasks or roller bottles, which typically provide about 850 cm2 of surface area on which cells can grow. "What is a mammalian cell-culture flask," asks Tim Ward, director of product management at The Automation Partnership (TAP) in Royston, UK. "It was not designed to make efficient use of its volume." And as an example, he adds that to build an industrial-sized machine equivalent to a 20,000 L bioreactor that relies on roller bottles "would require a facility the size of New York."
To address the problem, several companies are developing culture plates that increase the adhesive surface area within a given volume. Corning Life Sciences (Lowell, MA), for example, has developed several cell culture vessels meant to be used in high-volume cell production. Its "HYPERFlask," for example, provides a total of 1720 cm2 surface area on 10 growth layers within the footprint of one of the traditional T-flasks. According to Kim Titus, business development manager and the commercial lead for HYPERFlask vessel at Corning, the cells sit on a gas-permeable film through which all gas exchange takes place. Customers who have used the flask have verified that cells grow uniformly on each of the layers, indicating that nutrients and gases are distributed evenly throughout the flask.
Those considering even larger-scale production often turn to Corning's "CellCube Culture System." Each growth layer in a CellCube module provides 850 cm2 of surface area (the same area provided by one roller bottle) on as many as 100 growth layers for a total of 85,000 cm2 . The largest turnkey processes using the CellCube system right now uses four such 100-layer CellCube modules, but custom processes have been developed by some users to allow the use of up to 10 × 100-plate Cubes simultaneously, creating 850,000 cm2 of surface area for cell culture. Andrew Lesniak, applications specialist for bioprocess for CellCube at Corning, notes that the CellCube System, which was originally designed for bioprocessing those cells that could not go into suspension, can easily be adapted to large-scale culture of adherent cells. Culture medium and gases are pumped around the whole system, he notes, so that all of the cells in the cube have uniform access to them.
Providing adequate surface area on a reasonably-sized platform is just part of the challenge in considering large-scale cell culture. Many issues loom large when process developers consider how to automate the process in order to make it economically viable. When one considers all of the judgments that need be made in manual cell culture, it becomes clear that cell culture is more art than science. But it's that assumption says TAP's Ward that may have stymied development of automated cell-culture systems. By doing cell culture manually, "people find it difficult to get consistent results," he says. And that leads them to believe that cells are too finicky to culture industrially by machines that can't recognize every nuance of cell behavior and morphology. And that's where they make their mistake, says Ward. It's people's inconsistent handling of the cells that leads to inconsistent outcomes, he says. "If you can automate the process and treat cells consistently, you get consistent results. People's perception [of the challenge] is the reverse of reality."
The engineers at TAP, says Ward, "started out with some naiveté. Maybe if the team knew more about growing cells, we would have never attempted to do it." What TAP engineers built into their machines was consistency. "Automation makes cell culture more predictable. If you always treat cells in precisely the same way, you know exactly where they'll be in their life cycle at any point during the process, and the outcomes will be consistent." Ward notes that among TAP's collaborations is one with Manchester, UK-based Intercytex. According to an Intercytex press release, the two companies received a grant from the British government to "develop a dedicated robotic system to support the commercial-scale production of dermal papilla cells, the main cells involved in hair regeneration."
Even though the first generation of cell manufacturing is just starting to come on line, scientists like Rowley and Mason are looking to the future, asking how the field can get to RegenMed 2.0. Interestingly, both envision a process in the future where cell manufacture borrows even more from bioprocessing. The current paradigm, says Mason, mimics the manual procedures, but in the future, he imagines the emergence of a new paradigm. In one possible scenario, he imagines adherent cells on the surfaces of beads or wafers and then suspending the beads in bioreactors similar to the ones now being used for bioprocessing. This would truly allow enormous scale-up to bioprocess-type volumes. "That approach has not yet worked for primary stem cells," says Rowley. "But it is certainly an interesting possibility and may be a medium-to long-term goal of the industry," he says.
Of course as the cell-culturing process becomes more and more standardized, process developers will be confronted with challenges further down the manufacturing process. For example, cites Mason, stem cells are fragile and moving them into a container can exert shear forces that will tear them if you move them too quickly. "But if you go too slowly," he cautions, "they stick to the tube. There's a window of opportunity for moving them, and you have to hit it just right. There's a big investment in producing plastics that cells won't stick to." Ultimately, says Mason, the goal after the process is all standardized is "how to make it easier and cheaper to process cells."
1. C. Mason and M. Hoare, "Regenerative Medicine Bioprocessing: Building a Conceptual Framework Based on Early Studies," Tiss. Engin., 13 (2), 301–310 (2007).
2. L. Chue and D.K. Robinson, "Industrial Choices for Protein Production by Large-Scale Cell Culture," Current Opin. in Biotechnol., 12, 180–187 (2001).
3. C. Mason, "Regenerative Medicine: the Industry Comes of Age," Medic. Dev. Technol., 18 (2), 25–28 (2007).
4. The Automation Partnership, "Major Research Consortium Chooses CompacT SelecT for First European Study on Automating Cell and Tissue Production," Company Press Release, Nov. 22, 2005.