December 1, 2015 (Vol. 35, No. 21)
Angelo DePalma Ph.D. Writer GEN
You Don’t Need To Be a Viral-Clearance Guru
Virus removal and inactivation continue to loom large in overall bioprocess safety. As processes, products, and unit operations proliferate, viral clearance will become more customized to individual situations.
Simultaneously, viral clearance will keep broadening its purview. Initially, viral clearance focused on a small set of viruses, a set comprising viruses that were already identified and associated with the production of cell lines. Today, viral clearance encompasses unknown and uncharacterized agents. Moreover, as viral-clearance practices evolve, standards intended to decrease the risk of transmitting viruses will evolve, too.
Successive standards and guidances have already been issued by organizations such as the U.S. Food and Drug Administration (FDA), the European Medicines Agency (EMA), and the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH). The standards promulgated by these organizations illustrate the evolution of thinking with regard to virus safety.
Current standards reflect a holistic approach. According to Alison Armstrong, senior director of development services at BioReliance, regulators not only examine materials sourcing, but also assess manufacturing processes for virus-clearing capabilities.
Some manufacturers are implementing viral-clearance technologies in their upstream processes by including an inactivation or removal step for cell culture media. Inactivation may involve high-temperature short-time (HTST) treatment or ultraviolet-C (UV-C) irradiation, and removal may be accomplished by means of filtration. Such techniques have already demonstrated their usefulness in reducing the risk of viral contaminants in the manufacturing process. They may show even more efficacy in new configurations.
Capture, the affinity step immediately following harvest, is where virus removal begins in earnest. Shelly Parra, senior manager of global applications at Thermo Fisher Scientific, notes that bioprocessors often introduce intermediate or secondary washes while the product is still on the column. These washes can inactivate or cause viruses (and other contaminants) to flow through before elution. “We see a lot of inactivation during the protein A step due to the acidic environment of the elution,” Parra says.
Chemical inactivation is also possible, but Thermo Fisher’s specialty for viral clearance is anion-exchange chromatography, run as a second-stage column or as a polishing column. The company has an established resin, Poros™ HQ, which employs a 60% quaternarized polyethyleneimine functional group that imparts high salt tolerance.
“The ability to load onto these anion-exchange resins in flow-through mode at higher salt concentrations provides greater flexibility in terms of process continuity, particularly for continuous processing,” Parra notes. Poros HQ has been platformed for numerous virus removal steps in commercial and clinical manufacturing processes.
The more recently introduced Poros XQ, which has higher capacity than the HQ resin, is also a strong anion-exchange resin based on a 100% quaternary amine chemistry.
Not surprisingly, bioprocessors appreciate the opportunity to improve productivity of viral-clearance operations. In the past, anion-exchange columns had to be quite large to provide adequate throughput. Today, columns are smaller, and flow-throughput velocities are much higher.
“Conventional wisdom was to use a 20 cm bed running at 100 cm/hr to get good viral clearance,” Parra explains. “And that’s where a lot of the foundational work was done. Now processors are pushing the capabilities of resins and columns through faster flow rates and shorter bed heights.”
For example, a column based on Thermo Fisher’s HQ resin might only be 5 cm in height with a flow of 1,000 cm/hr. “That’s really fast,” exclaims. “You can use anion-exchange resins almost like a membrane.”
Anion-exchange membrane adsorbers are attractive because they are easy to deploy, but their utility drops off as scales increase. “Once a process scales up to commercial production,” Parra informs, “the membrane is replaced with a column.”
Biomanufacturers increasingly look for comprehensive virus-related services. “Our clients are requesting that our viral-clearance experts perform all aspects of the viral clearance study,” says Jeri Ann Boose, Ph.D., senior director of biopharmaceutical services at Eurofins Lancaster Laboratories.
Traditionally, Eurofins’ clients came to their testing lab and performed each step of the study with testing lab personnel assisting. Through the firm’s enhanced service offering, the client may still elect to come to the lab, but they may only perform the more complicated process steps such as column chromatography and allow lab personnel to perform the straightforward steps such as solution inactivation.
“At the extreme of this service, the client does not have to come to the testing lab at all,” notes Dr. Boose. “We instead perform a technology transfer of the scaled-down manufacturing process, and testing lab staff perform the study from beginning to end.” In some situations, the client may also request that the testing lab develop and validate the scaled-down process.
Eurofins also works with clients during the study-design stage to guarantee they will achieve clearance goals for each virus tested. First come detailed discussions on the purification process, followed by the presentation of multiple options for study performance based on knowledge of the ability of each step to clear individual viruses.
At present, viruses of special concern are the small, non-enveloped viruses. “These viruses are very difficult to inactivate and can pose a challenge to removal steps as well,” warns Dr. Boose. “Industry has therefore begun migrating to the use of HTST treatment and UV-C irradiation to inactivate them.”
Protein A columns provide a modest degree of viral clearance, so biomanufacturers often validate these columns as part of the clearance plan. A joint project involving Genentech and FDA’s Center for Drug Evaluation and Research examined viral-clearance performance on protein A based on company and agency historical data. Researchers found that for a given model virus, removal by protein A varied among different monoclonal antibody processes.
Interestingly, within a particular process, clearance was similar across the panel of test viruses. Also, it was determined that virus removal could be optimized through design of experiment.
Mapping the design space of a process step for viral clearance is the best way to optimize a manufacturing step for virus reduction. A design space may be developed in which optimal viral reduction and good product purification and recovery are balanced.
“These data lead to a good understanding of the mechanism of viral clearance, and of how it is influenced by the process step’s operating parameters,” explains Dr. Armstrong. “This can also become the basis of a platform approach to manufacturing.”
Depth filtration also could be tweaked to provide a measure of viral clearance. Both electrostatic and size exclusion effects are thought to be responsible.
Using a counter-ion displacement approach, a group at Eli Lilly led by senior research advisor Dayue Chen, Ph.D., tested the degree to which electrostatics facilitated removal. They tested two depth filters from EMD Millipore that differed significantly in ionic capacity. One filter, the highly ionic X0HC, completely cleared porcine parvovirus to below detection limits under low conductivity, achieving a log10 reduction factor greater than 4.8. The B1HC filter, with low ionic capacity, achieved a reduction value of less than 3.0.
The viral-clearance process for biosimilars is the same as for new products, so the same guidelines apply. Typically, a small-scale viral-clearance study is requested before the product enters clinical studies, and a more extended study is conducted for marketing authorization. But for biosimilars, at least, the time between Phase I studies and market authorization is much shorter than for new products, and the risk of product failure during clinical phase is also lower.
“This raises questions about the early- and late-stage viral-clearance approach,” observes Horst Ruppach, Ph.D., global director of viral clearance and virology, Biologics Testing Solutions, Charles River Laboratories. “In fact, companies producing biosimilars are now considering more extended viral-clearance studies at the early stage to reduce expenses and the time it takes to bring the product to market.”
Cell therapy is another rapidly growing area for viral clearance. Unfortunately, the production process frequently does not have viral-clearance capacity, which raises the risk of viral contamination. The viral safety of raw materials used in production therefore becomes more important because viral-clearance capacity of raw materials can significantly contribute to the overall virus safety of the cell therapy product.
The viral safety of recombinant products through viral clearance is typically addressed during downstream purification, where virus-retentive filtration is the most robust and effective viral-clearance step. With this in mind, many companies are investigating implementation of the virus filter upstream for media supply.
“Since the virus filter was designed for use in the downstream process, for filtration of nearly purified products, it will have to be adapted to the faster and higher total volume throughput required for media supply,” Dr. Ruppach asserts. “Virus-filter manufacturers are working on filters specifically designed for this use, and the first prototypes are currently being investigated.”
The biopharmaceutical industry is also investigating continuous processing, for which viral-clearance capacity has not yet been fully investigated. The demonstration of clearance in a downscaled model needs to be reconsidered for this approach. Ultimately, the newly developed virus-retentive filters for media supply might become the better virus filter to use in a downstream continuous purification process.
Regulatory agencies are beginning to acknowledge the results of the many studies archived in their databases and at biopharmaceutical and contract service organizations. “This discussion has just begun, though,” Dr. Ruppach cautions. “Not all agencies will accept such approaches,” at least not in their current form. “Some conclusions drawn from the databases will have to be carefully reviewed.”
For example, anion-exchange chromatography is thought to be a safe and robust viral-clearance step under specific pH and conductivity conditions. “This conclusion, however, is based on data derived from the same three or four model viruses,” Dr. Ruppach adds. “Studies have shown that if other models are analyzed, no or very low viral clearance is achieved under the same conditions.”
Sober Bioprocessors Spike Their Fluids
For many years, the virus spike was a bone of contention. For example, it wasn’t clear whether it should be introduced in a crude or a purified preparation.
Eventually, the conclusion was reached, says Jeri Ann Boose, Ph.D., senior director of biopharmaceutical services at Eurofins Lancaster Laboratories, that “different preparations should be used depending upon where the step being tested is in the overall product purification process.” Today, it is also known, Dr. Boose adds, that “the use of extremely high-titer stocks do not necessarily mean better clearance,” and that titers could even be high enough to introduce artifacts into study performance.”
The use of high-quality virus stocks is a key component to a successful viral-clearance study. Viral stocks should be propagated in low-serum or serum-free medium without indicator dyes. Also, in most cases, they should be subjected to a purification step and suspended in buffer.
“These stocks should be confirmed to be free of bacterial and mycoplasma contaminants and have certification to show high titer,” insists Alison Armstrong, senior director of development services at BioReliance. “Further purification and examination of protein, DNA, and RNA levels provides high-purity stocks that are essential for virus-reduction filtration studies.”
Still, many believe that pure spike virus is the way to go for demonstrating log-clearance to regulators. For example, EMD Millipore has exclusively licensed its TrueSpike™ technology to Charles River Laboratories for use on Charles River’s viral-clearance services. TrueSpike enables removal of cell debris, DNA, and nonviral proteins from virus preparations used to validate viral clearance.
The technology reduces the protein content of virus preparations used for validation studies up to 600-fold, as shown in studies with a TrueSpike minute mouse virus product. A significantly cleaner preparation can lead to increased predictability of validation studies, according to a statement released by EMD Millipore. TrueSpike also allows users to prepare higher concentrations of viruses, asserts Christophe Eyer, head of Provantage E2E Services at EMD Millipore.