April 1, 2005 (Vol. 25, No. 7)
Combining Tried and True Methods with New and Emerging Technologies
Amidst a shortage of new, robust viral clearance and validation technology, scientists are debating the relative advantages of viral inactivation versus viral clearance, and are considering new approaches to filter retention and new orthogonal clearance methods that may combine the tried and true with the few new and emerging viral clearance or inactivation technologies.
This field, often described as static, changes in concert with biopharmaceutical process development. Lately, however, it’s getting a bumpnot yet a surgeof interest as regulatory bodies begin to look more stringently at drug development and consumers scrutinize the industry.
“In principle, the risk of viral contamination is a feature common to all biotechnology products derived from cell lines and human or animal plasma,” notes Klaus Tarrach, senior product manager, purification technologies, Sartorius (www.sartorius.com).
“According to recent publications, no infections or transmissions of CHO cell-related type A or C virus particles to other cells have been reported, but retrovirus-like particles theoretically can pose safety concerns due to their similarity to tumorigenic retroviruses.”
“Size exclusion filters were an early way to add viral clearance into a process,” says Jerold Martin, senior vp and global technical director, Pall Life Sciences (www.pall.com). Now, orthogonal methods are used routinely, incorporating virus filtration, inactivation, and adsorption technologies, sometimes measured by Q-PCR.
“They comprise individual technology platforms where each removal or inactivation step contributes significantly to the overall virus clearance capability of the process,” Tarrach adds.
“Virus filtration and inactivation traditionally have been accepted as complementary and robust methods for viral clearance,” he continues, and chromatography is a popular method of removing viruses, regardless of size or morphology.
“However, chromatographic removal is contingent upon the chemical composition of the mobile phase and iso-electric point of each virus species,” Tarrach notes.
Ion Exchange Membranes
In the past few years, ion exchange membranes have been applied to the validation challenge. “Ion exchange membranes with a positive surface charge offer an advantage over chromatography beads,” Martin says.
“Traditional chromatography resin beads have pores suitable for protein purification but are too small to effectively remove DNA and other large biomolecules. Virus particles can’t readily penetrate the pores of beads, so beads have low capacity.” Therefore, much of the capability of a standard chromatography column is underutilized, requiring oversized columns in final contaminant removal applications.
In contrast, a microporous ion exchange membrane device increases virus access to adsorptive surfaces and thus increases virus removal and direct flow rates.
“Interest in membrane chromatography as a polishing step for final DNA, host cell protein, and viruses is new,” Martin says.
Within the filtration industry, it’s difficult to compare filters directly because of the different nomenclature systems used by manufacturers. The PDA has formed a viral filtration task force to establish a standard for naming and rating virus filters so each manufacturer may provide grade descriptions using the same nomenclature.
“New developments in virus filtration are targeting higher area filters, which provide increased flow rates and throughputs,” Martin continues. Pall’s new Ultipor VF UDV20 filters, for example, offer twice the filter area in a 10 modular cartridge.
“The patented Ultipleat pleating technology lets us pack more membrane (2 square meters) in the cartridge.” This size exclusion filtration method isn’t affected by minor changes in process parameters and, he says, “is resistant to plugging and flux decline, even during spiking studies.”
The next generation of filters will be highly efficient for even small 20-nm viruses, while still allowing a 95% transmission rate for 160 kDalton and smaller molecules at a practical flow rate.
Virus Spiking Studies
Millipore (www.millipore.com) is working with the FDA and its customers to improve methods to validate the effectiveness of the viral clearance step. One promising method of performing virus spiking studies could expand filter life and dramatically reduce viral filtration consumable costs by up to 75%. The benefits are based less on technological change than upon changing perspectives.
Spiking studies are performed at lab scale using process intermediates and virus stock preparations, which are prepared by cell culture methods and may contain a variety of impurities.
During spiking studies the filters may become plugged by impurities “atypical from the actual process stream,” according to Mani Krishnan, technology manager, virus management solutions at Millipore. Currently, “capacity obtained in small-scale studies defines the filtration endpoint, despite the fact that the process fluid alone may cause significantly less plugging.
“Based upon our understanding of how filters plug, we recommend using flow decline, rather than capacity, as an endpoint,” Krishnan says. “The difference is that customers currently are throwing away filters earlier than needed,” notes Glen Bolton, Ph.D., application R&D manager, Millipore.
“There’s growing acceptance within the industry that viral filters can be added at the end of the process for enhanced polishing. As more drugs move into late-stage clinical trials, you’ll see more use of the flow decline method,” Krishnan predicts.
One of the challenges, however, is that the viruses and the products often are of similar size, making size-exclusion methodologies less effective. For example, Tarrach elaborates, “Porcine parvo virus poses the biggest challenge for all viral clearance studies, as this virus is the most difficult to inactivate or remove. Plasma manufacturers targeting factor VIII or other related products need a virus clearance strategy with 12 to 20 log clearance.
“A 20-nm virus filtration system is economically not indicated, as the products will be significantly retained by the virus filter itself. Therefore, virus clearance strategies must consider other removal or inactivation technologies.”
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.”
Cleaning Validation Studies
Its not just downstream processes that need virus validation studies. A trend noted by BioReliance (www.bioreliance.com) is an increasing demand for cleaning validation studies. These studies are performed to validate decontamination and sanitization procedures for the treatment of surfaces, chromatographic resins, and instruments that come into contact with biological products.
The studies analyze the suitability of selected cleaning agents to inactivate or remove contaminants such as viruses, bacteria, fungi, mycoplasma, or prions. Decontamination procedures, particularly those used between batch runs of biological products, are critical in order to avoid costly contamination events, which could result in the loss of product.
Other validation studies that BioReliance is being asked to perform relate to the re-use of chromatography columns. For example, carryover studies can be used to assess the ability of a manufacturing process or sanitization procedure to remove or inactivate contaminants on recycled chromatography columns. Viral clearance studies can also be used to measure the ability of re-used column resins to clear viruses after multiple runs.