Filtration with viral removal filters is a traditional clearance step, and most manufacturers have been able to optimize their product for filtration without any significant loss of product. “However, when the process undergoes the actual virus clearance studies, and viruses are spiked into the product in a scaled-down study, the performance of the process step can sometimes be problematic,” according to Joe Hughes, Ph.D., vp biologics testing, WuXi AppTec. “Between 90 and 95 percent of the time, filters run as expected,” he reported.
For that remaining 5 to 10%, the quality of the virus spike often causes the filters to plug. To address this situation, WuXi AppTec is advancing two technologies to prepare virus for virus removal studies—ultracentrifugation for enveloped viruses and chromatography using membrane absorbers for nonenveloped viruses. Dr. Hughes will discuss WuXi’s approach in detail at Informa Life Science’s “Viral Safety for Biologica” conference in Cologne next month.
Ultracentrifugation involves putting glycerol, which acts like a filter, on the bottom of the centrifugation tube. “The virus pellets through the glycerol,” he said, “resulting in a cleaner virus preparation, but holding back much of the cellular proteins and membrane fragments.” WuXi’s version is known as Ultra 1, and routinely results in a five to seven log viral removal, without reducing flux, reported Dr. Hughes. It’s not a good fit for all viruses, however.
Therefore, WuXi also offers Ultra 2, which is designed for parvovirus and for minute virus of mice. In quality control testing, compared to Ultra 1, “Ultra 2 takes away three- to fivefold more protein and 100-fold more nucleic acid,” Dr. Hughes noted. “Performance of the Ultra 2 virus preparations on virus removal filters has demonstrated good removal of the parvoviruses, often without any changes in the product filtration performance.”
For the parvovirus purification, an anion exchange based membrane absorber is used. The virus will bind to the absorber in a low salt solution and can be eluted with 0.4 M sodium chloride solution, but the nucleic acid will be strongly bound and is essentially removed from the resulting Ultra 2 virus preparation.
The result, Dr. Hughes says, is that when the virus is spiked, “it doesn’t clog the filter.” This method is most effective for parvovirus and other nonenveloped viruses. Researchers are looking at this method for other nonenveloped viruses, as well as other matrices as a way to expand its applications. “All of these enhancements should allow manufacturers to effectively validate their filters, and perhaps even reduce the size and overall cost of the filters needed in the process.”
Millipore launched the Viresolve® Pro family in several phases in 2008, and the FlexReady platform in March 2009, as a virus-clearance solution to remove parvovirus from mammalian or recombinant sources. The technology, which was described at the Dusseldorf conference, features a patent-pending membrane, a newly designed device format, and more sensitive performance tests.
“The design of the membrane is based on worldwide feedback from our customers,” explained Gerd Kern, field marketing manager, bioprocess division. The result of their feedback is a membrane that helps companies ensure regulatory compliance and manage their business risks associated with virus safety, he explained.
The dual-layer PES membrane is designed to simultaneously provide high retention, high capacity, and high flux. This is important, Kern said, because there is a trend to compress the chromatography steps before this purification step from three to two, leading to a greater impurity load at this point. “It processes fast,” he added, “and is caustic stable, so it can be sanitized with everything in line. This is a big leap forward in terms of overall performance.”
The new device format scales from development through manufacturing and integrates easily into the new Mobius FlexReady platform, Kern noted. The Viresolve membrane is available in Micro, Modus®, and Magnus® formats, which are designed to fit in all processes and enable productivity gains in all production scales.
“Viresolve Pro has a fully disposable flow path, so there’s no cleaning or cleaning validation,” Kern said. More robust performance tests including binary gas tests also have been developed “to detect the smallest defect and to confirm virus retention confirm.” The Viresolve Pro has been designed to remove Ž4 logs of parvovirus and Ž5 logs of retrovirus. Versions of the Viresolve Pro are available for process development and virus validation, the Modus for pilot scale and medium processing, the Magnus for large-scale processing by using the Viresolve Pro holder.