The industry is evaluating the capability of various manufacturing processes to remove proteinaceous infectious particles (prions) from the feed stream, Tarrach says.
"Prion clearance validation studies are necessary if, during manufacture, the product contacts animal or human tissues that could be infected with a transmissible spongiform encephalopathy (TSE) agent." Such studies, Tarrach says, "generally feature Western blot analysis for initial investigation," which helps determine effective procedures and technologies.
"Once determined, these tests must be followed by in vivo studies in mouse or hamster lines if there is no data available showing a correlation between the Western blot analysis and existing in vivo studies related to this specific clearance technology.
"One of the latest viral clearance technologies is virus inactivation by UVC light, featuring a novel hydraulic spiral flow UVC inactivation module from Bayer Technology Services (www.bayer.com)," Tarrach says. "We integrated this new generation of continuous-flow UVC reactors, operating at 254 nm, in our virus clearance technology platform," which Sartorius and Bayer developed jointly.
"In the UVC reactor, a novel hydraulic spiral flow along an irradiation source generates Dean vortices in a fluid stream that provides homogeneous residence time distribution of the product. This allows the doses of UVC irradiation to be delivered uniformly throughout the solution.
UVC treatment is accurately controllable and provides the end-user with a precise window of operation. Reliable 4 log titer reduction of porcine parvo virus in biopharmaceutical feed streams have been shown recently," Tarrach says.
As the final step in Sartorius' three-step viral clearance technology platform, it introduced the Virosart CPV membrane filter. "Each of the three orthogonal viral clearance technologies in this platform target virus and prion clearance, feature clearance of porcine parvo virus of more than 4 log reduction, and act independently."
At Covance (www.covance.com), "Our key message to clients is that early planning for viral validation is vitally important," notes Carl Martin, Ph.D., vp, biotechnology.
"Fortunately, the old days of designing the purification steps with only the product in mind, and viral clearance as an afterthought, are gone. Regulators want one, and preferably two, robust steps, each showing a four to five log viral clearance. Any changes to the process can be very costly, so it's important to get it right the first time."
Companies often don't provide enough data for scale down. In fact, a German Federal Department of Health representative noted that validation of process scaledown was the major area where regulators receive insufficient data, Dr. Martin says.
"There's a lot more to viral validation than many companies realize," he adds. For example, column buffers and inactivation agents can cause toxicity to cells and interfere with virus infectivity.
Hence, detailed cytotoxicity studies and virus interference assays both should be conducted before the main validation study to ensure successful viral assay during the clearance studies. Yet, Dr. Martin adds, "Companies often haven't budgeted for this."
Additionally, Dr. Martin says, some regulatory bodies tend to prefer inactivation to removal, as inactivation is easier to demonstrate. They argue that removal methodologies are more prone to failures caused by changes in buffers, leading to large differences in partitioning or virus elution.
"Enveloped viruses can, in general, be cleared. It is the non-enveloped viruses that are problematic," he says. "Presently, the smallest of these are best removed by inactivation methods which, unfortunately, can be deleterious to the product."
Viral validation is based upon spiking studies using a set of relevant viruses. "From time to time, that set is changed," says James Gilbert, Ph.D., senior director, biopharmaceuticals, MDS PharmaServices (www. mdsinc.com), as when West Nile virus was added in the U.S. a few years ago.
At Navigant Consulting (www.navigantconsulting.com), "We've seen some companies double the list of viruses," notes Eric Manning, Ph.D., senior consultant.
The issue Dr. Gilbert sees most often is that "the process isn't robust enough, so the proteins used can't withstand the removal or inactivation methods needed."
Additional challenges, he says, are designing a spiking recovery study for completely new products for which there are few references. Occasionally, logistics must be examined, too, as certain steps of a production process may be conducted at distant sites that necessitate sample stabilization and an understanding of the conditions under which the sample was shipped.
The key to successful viral clearance and validation, Dr. Martin says, "is to look at both the virus inactivation and the integrity of the product itself, conducting a detailed analysis of the combined data."