Improvements in technology and media spur advances, but automation also creates new challenges in culture processes
Cell culture plays a vital role at every stage of the pharmaceutical and biotechnology industry, from target identification, where smaller numbers of cells are needed,
through manufacturing, which requires vast numbers of cells. But while cell cultures are ubiquitous, they are not without problems. In research labs around the world, scientists wrestle with challenges that vary depending on which stage of the research and development pipeline they are at. In addition to grappling with expression levels, variability in cell lines, or maintaining sterility, researchers also have to face the tedium that often comes with traditional manual cell culture methods.
“Cell culture is something where people have to be very observant and very careful and very clean. It’s quite demanding technically, but it’s also very time consuming and very repetitive. People need to have a really high attention span, the tolerance for the same thing over and over, and often it’s difficult to get a good sense of reward for people who are doing work like that,” says Alison Rush, PhD, senior research associate, department of automated biotechnology, Merck Research Laboratories, North Wales, Pa.
Rush is lucky enough to be working in an automated cell culture environment, but that can come with its own set of troubles. There are many different assays used, including development assays, high-content assays, and follow-up assays, which require many different types of cells in medium quantities. Ultra high-throughput screening also requires billions of cells.
Another problem facing the field is improving cell culture media, says Laurie Donahue, PhD, manager of technical development for Sigma-Aldrich Corp., St. Louis. Although everyone recognizes standard well-known media components that need to be optimized in a formulation, researchers in the field are now investigating novel components. It is common knowledge which amino acids, which vitamins, which trace metals need to be examined or optimized or perhaps even left out of a formulation, but it is unclear what role signal transduction pathways, cell cycle control, and metabolomics play in testing novel media components.
“If we’re trying to block the cells from dying in the bioreactor due to apoptosis, have we really explored the entire apoptotic pathway to know perhaps if we should add a particular caspase,” says Donahue. “We’re at an interesting edge in development where everyone knows all the basics and now we’re all trying to create and find new ways to improve the medium using all this new knowledge that’s come out of the basic science labs. But that has not come to fruition; people are exploring it.”
Improving process efficiency
Zhijian Lu, PhD, is the associate director for biological technologies at the Wyeth Discovery Campus, Cambridge, Mass. His group provides protein drug candidates in
|Giving Cell Culture Media a Second Look
Like many tools used in laboratories on a daily basis, cell culture media often do not get a second look from researchers. But while they are often perceived as just another tool, there is a lot more to them than people realize, says Laurie Donahue, PhD, manager of technical development for Sigma-Aldrich Corp., St. Louis. “Cell culture media are more complex than most people who don’t work in that industry realize.” For example, two research labs producing antibodies using Chinese hamster ovary (CHO) cells derived from the same parental CHO line might be surprised to find out how different they are.
“Those two lines, even though they come from the same parental CHO, will have completely different nutritional requirements. Yes, they’ll all need amino acids and hormones and vitamins and trace metals, but the exact concentrations and the relative concentrations will be different, so it’s a big challenge to find the right medium,” she says. And although researchers may successfully use a particular media formulation for one cell culture situation, it may not work for another. “It’s a frustration for the pharmaceutical industry because they hope to find one medium that might be a great platform that they can always use with their lines, but it just doesn’t seem to work…,It means we just don’t understand something fundamental about the physiology of these cells.”
Sylvia Hu, PhD, director of research in the department of protein sciences at Amgen Inc., Thousand Oaks, Calif., says that for general research applications such as early stage target validation, her labs tend to use standard commercial media. Which one depends on the cell line they are working with, so they will conduct a comparative investigation to determine the optimal media.
a form that can be used in animal experiments, and they also provide cell culture support for high-throughput screening in small-molecule drug discovery. “On a day-to-day basis, we do a very heavy amount of work in cell cultures, mainly with a lot of mammalian cells. We also have purification work going on to purify proteins generated from our cell culture processes,” Lu says.
Wyeth has been advancing 12 molecules annually into development, typically nine small molecules and three proteins. They have achieved that by increasing process efficiency without significantly increasing hours. One tool that played a part in this increased efficiency was the Wave Bioreactor from Wave Biotech, Bridgewater, N.J., a cell culture system that can handle up to 500 L. It features a sterile, disposable chamber called a cell bag that is placed on a special rocking platform that induces waves in the culture fluid. “They really fit our needs very nicely and we can switch from cell line to cell line very quickly without concern about the process,” says Lu.
Another factor in their increased efficiency was a preadapted Chinese hamster ovary (CHO) cell line developed in house that begins to grow in culture in a matter of days. Before the preadapted CHO cell line was available, growing CHO cells in culture could take four to six months. “These have become widely recognized over the last few years as a very useful tool to improve the process.” Wyeth’s efforts to develop these cell lines go back nearly a decade, and other companies have developed their own preadapted CHO cell lines, Lu says.
His group also benefited from work done by colleagues in Wyeth’s development and manufacturing group for protein technologies, and much of that work has increased productivity of mammalian cell culture to very high levels. This was achieved by improving methods to select among hundreds of clones to find the one that best met production requirements for stability. There was also media engineering so that very high cell density could be achieved from the batch culture.
“By engineering the metabolism of cells, the waste generation can now be overcome so that the long-term culture process becomes feasible,” Lu says. “All of these incremental improvements permeate into discovery. We try to adopt as many of these advances as possible. We can also achieve a higher level of expression, and this will benefit the overall process, so we can go through campaigns very quickly to make the amount of protein needed for in vivo experiments.” Despite those advances, he says that his biggest challenge on a daily basis is protein expression levels, because every protein behaves differently in cell culture. Expression levels are more of an issue in early discovery because there are multiple versions of clones to examine.
Generating cell lines
The focus of the lab headed by Sylvia Hu, PhD, has widened from primarily developing cell lines for production or manufacturing to generating cell lines for small-molecule library screening at Amgen Inc., Thousand Oaks, Calif. “The skills and knowledge that we learned in cell culture to support production can often be applied to generate better and more efficient cell lines for high-throughput screening, target validation, and other things,” says Hu, director of research in the department of protein sciences.
The difficulties she faces come at the discovery or target validation phases, where cell lines are much more variable due to inadequate tissue culture media. “It’s much more labor intensive and time consuming. The worker often has to work with multiple cell lines, even some with different behavior, requiring different processes and media and applications. It’s not possible to fit them into a standard generic platform for producing cell lines.” Hu’s lab gets support downstream from scientists in process development and assay development that allows them to look at product quality at early stages. “I think that part is fairly mature and all the components are in place.”
Another daily challenge researchers face is working with hybridoma cells, which are often used in industry and academia as an important starting point for antibody production. Hu says the production media for hybridoma is years behind those used for standard CHO and other production cell lines. “That is an area where we would like to see continued development.” In addition, when working with hybridoma cell lines, researchers must quickly evaluate thousands of individual hybridoma clones to analyze their productivity and antibody production, something that Hu describes as very labor intensive and stressful. “[Development of] a successful way to either automate some of those processes or to make a decision on clone selection at very early stages of the hybridoma work will have a tremendous impact on helping out these early-stage projects.”
Automation has affected some aspects of cell culturing, and Merck’s Rush has experienced it firsthand. She is managing a group that includes two other people and has
access to SelecT, an automated cell culture system from The Automation Partnership (TAP), Royston, UK. “It was a big shift automating because people who do cell cultures typically come from a research-based background and generally aren’t all that familiar with automation. The introduction of a robot to do that requires a whole new skill set. One of the significant challenges is finding someone who can handle both the cell biology and the automation at the same time. Typically those two groups of people have been in different environments,” says Rush.
In a high-throughput environment, researchers need to become familiar with both the automation hardware and software, and one way to do that is by experimenting with the robot and the science. Although it is easy for scientists to toy with traditional cell culture techniques to tweak them, that is not so easily done with automated techniques. “Now things have to be done in a very ordered way. It’s a different way of thinking about experiments,” Rush says.
Automation has changed the face of cell culturing, but there are still some areas that are under-served. One example is some of the more complex plate-based assays where researchers need to do feeding or additions, and which require growth in the presence of different compounds. Rush says that Cello, TAP’s automated system for mammalian cell culture in plates, could fill that role. The development of other technology could help researchers deal with the difficulties that arise when trying to perform ultra high-throughput screening requiring huge numbers of cells for a wide variety of cell-based assays. Tecan AG, Zurich, Switzerland, has developed Cellerity, an automated cell culture system that can be used for cell-line maintenance, expansion, harvesting, and plating.
Donahue from Sigma-Aldrich believes the development of better tools will allow researchers to use more primary cells in their screens. “In many cases they’re using
transformed cell lines, continuous cell lines, to do studies that would be better done with a primary cell type that has a finite life span. But the ability to manipulate those cells is limited, not only in the number of population doublings the cell has, but in how little we know as to how to culture them.”
Two other challenges that researchers face are a lack of space in their labs and maintaining stability during cell culturing. Corning Inc., Corning, N.Y., recently introduced CellStack Culture Chambers for cell culture scale-up, protein production, and cell-based assays, and the company believes it addresses both these issues. “This has a 40-layer cell stack, so that’s one culture vessel that has the equivalent of about 30 roller bottles,” says Todd Upton, PhD, applications manager for cells at Corning. “That’s 30 times less opportunity for contamination, 30 fewer times you have to manipulate the vessel to grow cells, change media, or harvest cells.” CellStack is also a closed systems that can be manipulated by the use of multiple entry and exit ports that can be used without breaking sterility.
Filed Under: Drug Discovery