Editorial Article
Monday May 24, 2010
by Josh P. Roberts
It's one thing to work in a research lab: cobbling funds together from research grants, conducting experiments in 96-well plates, using stand-alone benchtop equipment, and meticulously tweaking reagent concentrations, incubation times, and column densities.
But as R turns to D and it's time to scale up and start producing product, a host of other considerations come into play. Some of these—like streamlining a process, allowing for flexibility, or maximizing return on investment—may be front and center in the minds those involved in bioprocess. Others, perhaps just as important, may not be so obvious.
Bioprocessing is often, at least mentally, divided into two phases: upstream or fermentation, and downstream or purification. Perhaps the biggest upstream issue that Fred Schendel, who leads the fermentation side of the University of Minnesota's Biotechnology Resource Center, sees, is integrating data collection with fermentation.
Downstream there tends to be a trend toward single-use, especially in the development and small scale manufacturing stages. Integrating downstream with upstream is becoming more important, and with it collaborations among users and multiple vendors who supply them, leading more toward a one-stop shopping for a bioprocess.
And finally, monitoring not just the process but the stability of the product is important for a host of reasons as well.
What's in there?
Having a consistent process and product—whether it's beer or biopharmaceuticals—requires that fermentation conditions be both controlled and monitored. Too much glucose in the mix, or the wrong oxygen concentration, for example, and the culture may consume too much media, or utilize a different set of pathways, generating toxic by-products or those the downstream steps aren't designed for.
Monitoring can be done by versatile, tried-and-true methods like HPLC. But these typically require a large upfront investment and a very skilled operator, argues Sara Veth, technical marketing consultant to YSI Life Sciences. Rather, YSI and other vendors offer instruments that will periodically sample and monitor various nutrients and byproducts directly from a bioreactor. Results can be sent to a printer or a PC. Data can be integrated with controls that help keep the system automatically operating within specified parameters.
“We can set it up so you can do online monitoring,” adds YSI Director of Sales Steve Grant. “There's even an application to run it off an iPad or an iPhone.”
Reduce, but don't reuse or recycle
Even as the fermentation process is modernizing, much of the downstream side of bioprocessing is still a bottleneck, observes Maik Jornitz, group vice president of marketing and product management for Sartorius Stedim
North America. But it is being addressed to a certain degree in the ion exchange polishing and purification steps. “For example, a few years ago one had to use the classical gel chromatography within the polishing step.” he says. “Nowadays there is an increasing interest toward membrane chromatography—these membranes run, by far, faster than the classical chromatography column, [and] the buffer requirement for membrane chromatography is at least five times lower than with a typical resin chromatography.”
Perhaps counter-intuitively, another way the field is reducing waste is by turning to single-use technologies. Although single-use components require energy and raw materials to manufacture, and spent products need to be disposed of, this is more than offset by the savings in WFI (water for injection) usage for cleaning and sterilizing—resulting also in decreased energy consumption—and many other environmental savings.
Jornitz extols the virtues of single-use technologies far beyond their greenness. For example, the capital investment required is far lower: “You don’t have to buy a 1000- or 2000-liter stainless steel bioreactor, which takes around 2-3 years to commission” he points out. Instead, a single-use bioreactor can be commissioned within seven months, and because there's no cleaning involved, there's no cleaning validation involved either. Similar benefits are valid for media and buffer prep, as well as for cell harvest. Single-use technologies are more flexible, manpower costs are reduced, the risk of human exposure is minimized, and even the footprint required can be curtailed.
Christopher Stenland, who is in charge of downstream processing at the U of Minnesota's BRC, points out that as a core facility, “we prefer single-use components.” They need to be hyper-cognizant of issues such as accountability and validation, and cannot run the risk of cross-contamination inherent in a multi-product facility.
Jornitz acknowledges that there are drawbacks to single-use technologies. It's important to check the compatibility of the polymeric equipment against what it's being asked to do, he cautions. What are the temperature limits? What kind of mechanical stability does it have? Will the polymer itself interact with the process (in terms of extractables and leachables)? “But that’s typical process qualification.”
Pieces of a puzzle
Jornitz looks forward to the day when process qualification begins with media formulation: “We should think ahead and ask, 'What happens when I put this cell culture component into my cell culture? What happens on the downstream side?' There are wonderful components to get a great expression rate, but then you foul up your purification step and you lose all the yield that you created in your cell culture area,” he says. “I think we have to look in the future into which we develop a media and a cell line or cell expression rate immediately together with the next steps of cell harvest and purification.”
One way toward such integration is for customers to collaborate with vendors, beginning at the small-scale production stage or even the lab bench. YSI will “typically start to see customers in R&D,” says Veth. “And typically the instrument would be moved with whatever product they're trying to manufacture through bioreactor optimization, and sometimes in to manufacturing—depending on the company.”
Vendors will also collaborate with each other. Filtration and liquid handling equipment manufacturer Sartorius Stedim Biotech, for example, recently partnered with SAFC Bioscience, the cell culture product division of Sigma-Aldrich, to “better serve customers in solution creation, validation support, process improvement, technical support and problem solving,” according to a press release announcing the partnership.
In the recent past such collaborations were usually initiated by companies partnering with each other and then selling the enhanced offerings to their customers. But customers have become more educated, and now often reach out to vendors to put together a complete package, explains Grant. “From there you go out and start uncovering who can help you with this. You may not even need to deal with something formally—it may be something where you can partner up with the company as separate entities by delivering an entire package.”
The vendor acts almost like a broker. “Call it consultative selling,” Grant continues. “You don't just sell a product. You're looking at an entire process and you're trying to build something to help with that process.”
Testing, testing
Sometimes that process needs to include ways of making sure the product behaves like it's supposed to, both along the way as well as in its final form.
According to Wayne Patton, CSO of Enzo Life Sciences, protein aggregation is the most significant obstacle to developing a protein-based pharmaceutical. It can lead to low yield, poor storage capacity, and increased production costs. The FDA is concerned that aggregation may make the drug more immunogenic and lead to other complications such as pulmonary embolism.
Standard methods for aggregate testing—such as size-exclusion chromatography, sedimentation velocity ultracentrifugation, and optical methods such as dynamic light scattering—tend to be very instrument-intensive and low-throughput. To address this, Enzo has developed rapid tests that make use of fluorescent dyes recognizing aggregated proteins. ProteoStat Protein Aggregation Assay looks at the amount of aggregate in a sample. A second offering, ProteoStat Thermoshift Stability Assay, is “an accelerated shelf life stability-type of test” that exposes a protein sample to a temperature gradient and asks at what temperature does it aggregate, Patton explains. By incorporating tests such as these, different conditions at various stages in the development process, as well as different protein variants, can be evaluated against each other.