By Angelo DePalma, Ph.D.
The significance of viral safety is apparent throughout the biopharmaceutical production process. The ultimate goal is to protect patients from pathogenic viruses, and biopharmaceutical manufacturers need to demonstrate viral safety and validate viral clearance capability of the manufacturing process before market approval.
Viral safety is critically important in both upstream and downstream processes. Factors to consider in upstream processes include: choice of expression system, degree and type of genetic manipulation of those cells or organisms, and how the cell culture is run. All approaches are selected to efficiently produce the biotherapeutic product while minimizing the possibility of virus entry into the system. In downstream purification, virus removal or inactivation is accomplished by a combination of orthogonal or complementary approaches that include chromatography, chemical inactivation, and filtration.
For this article, we turn to four experts from MilliporeSigma, all with unique perspectives on the various technologies that assure that biotherapeutic protein products not only comply with requirements for cGMPs and expectations of regulators, but ultimately provide the highest level of patient safety.
A mAb downstream suite. The mAb downstream viral clearance is accomplished by a combination of orthogonal or complementary approaches including filtration, chromatography, and chemical inactivation.
Adventitious viruses can enter the production processes from multiple different routes: from cells, raw materials, personnel, or the environment. In addition, mammalian expression cells contain endogenous viruses. “These viruses are known, quantifiable, and represent fixed-input virus levels that establish a baseline demand for removal or inactivation,” explains David Beattie, Ph.D., Head of Bioprocessing R&D, MilliporeSigma. Virus titers vary according to cell type, transfection methods, and expressed protein. For example, the NS0 cell line expresses higher virus titers than the CHO cell line, as do expression systems that produce cytokines. Thus, the inherent variability and uniqueness in addressing viral safety, according to Dr. Beattie, is “having a variable level of input virus that defines a baseline demand for clearance means your virus removal might be very effective in one situation, but inadequate in others.”
Using well-characterized cell lines, such as CHO, the preferred expression system for monoclonal antibodies (mAbs), is one way to reduce inherent virus loads, assuming the expression system is appropriate for achieving desired product yield and quality.
The second significant source of virus contamination are media components derived from animal sources. This risk, along with the fear of prion contamination, has been the major impetus for adopting animal-free or chemically defined raw materials. Even then, rodent infestations in plants from which raw materials are sourced can result in completely unanticipated viral contamination. “Genentech and Merrimack both experienced bioreactor contaminations by Minute Virus of Mice (MVM), a virus associated with mice,” notes Dr. Beattie. “Such adventitious contaminations can result in viruses entering the downstream purification train.”
Standard mAb Purification Platform
The standard mAb purification platform includes protein A capture, where significant viral clearance occurs, followed by one or more chromatography steps. Anion exchange chromatography “polishes” process streams of host cell protein and nucleic acid, and can be an important step for viral clearance. Cation-exchange chromatography, which removes process-related impurities like aggregates and charge variants, provides one to three logs of virus removal, which is modest but not insignificant. Viral clearance numbers are logarithmic, so three logs reduction is equivalent to a thousand-fold reduction in virus levels.
However, “if you determine on the basis of product yield or quality that you don’t need that process step, you lose its associated clearance,” Dr. Beattie points out. “The desire to trim downstream processing to simplify and enhance their productivity carries the risk of eliminating or modifying unit operations, thereby reducing or losing their capacity to remove viruses.”
Critical steps in the downstream process are those dedicated to viral clearance, including low pH and/or detergent treatment to reduce levels of enveloped viruses, and virus filtration, which removes both enveloped and nonenveloped viruses. Most processes rely on these dedicated steps to make major contributions to overall viral clearance targets. However, the impact of unit operations on the properties of the molecule can be quite complex. Low pH hold is highly effective for inactivating viruses but is hard on therapeutic proteins and may affect yield, highlighting the interplay of product-quality assessments with requirements for viral clearance throughout downstream purification.
Similarly, upstream-processing conditions will have a direct effect on performance of the downstream unit operations, and higher cell densities and volumetric productivity will also likely affect the amount of virus entering the purification train. These changes can all impact the efficiency of the purification operations for both impurity removal and virus reduction.
Establishing and conducting viral clearance testing for biopharmaceutical customers is the focus of Kathryn Remington, Ph.D., principal scientist focusing on the BioReliance® portfolio of MilliporeSigma. At a previous job at a large biopharmaceutical company, Dr. Remington collaborated with in-house process-development groups to build viral safety into processes from an early stage, and then evaluated the viral clearance potential of the manufacturing process. Today, services related to viral clearance are largely outsourced, as the time and cost of a dedicated viral safety group and laboratories are beyond the resources of most companies.
A process-centered view of viral safety makes sense since downstream unit operations serve as a “safety net” to clear any adventitious virus that might escape upstream testing. But Dr. Remington cautions that “while some downstream steps provide very good clearance, some don’t provide any at all. The level of clearance is process-specific, moleculespecific, and even virus-specific.”
A scientist developing a chromatography step at bench-top scale.
Some measures, like chemical inactivation through detergents, inactivate broad classes of viruses, as for example, lipid-enveloped viruses. Low-pH inactivation provides good inactivation of enveloped viruses. But, as mentioned earlier, not all products are stable under acidic conditions. As the pH increases above pH 3.5, inactivation of enveloped viruses becomes less robust. Chromatography clears viruses based on their interaction with the resin. “Each virus has its own isoelectric point and other physical characteristics that make its interaction with resins unique,” Dr. Remington says. Removal of virus by filtration is based on size, and high levels of both enveloped and non-enveloped virus can generally be expected to be removed.
Ideally, process developers build in sufficient steps to remove or inactivate as many potential virus threats as possible. “The overall strategy should aim broadly because we don’t know a priori what viral contaminants we may encounter,” says Dr. Remington.
The same step may not always provide the same level of clearance from process to process. “People believe that if a column works great at a certain pH for one molecule that it will provide the same level of clearance in other instances. But that doesn’t always work out,” she continues. “Sometimes the conductivity or the virus’ isoelectric point is not right.”
Given appropriate resources, optimizing purification, recovery, and viral clearance is possible. However, development groups typically focus on the first two objectives, then work clearance in afterwards by adding or enhancing certain steps.
“It helps to have sufficient resources to conduct feasibility or even a design of experiment study, to understand the viral clearance potential within certain ranges of operating parameters,” Dr. Remington adds. “This will provide greater confidence in implementing future process steps, especially if a platform approach is involved.”
An operator setting up a virus filtration step. Virus filtration removes both
enveloped and non-enveloped viruses.
Viral safety often depends on dedicated inactivation and removal steps, and in good part on downstream-chromatography operations with inherent viral clearing capabilities. As biomanufacturers squeeze processes for even greater productivity they must examine if those process improvements affect virus clearance of the individual unit operations.
Michael Phillips, Ph.D., director of next-generation processing R&D, MilliporeSigma, notes that three levels of process intensification could affect viral safety. These are particularly salient for CHO-based mAb manufacturing, where many new ideas in bioprocessing are first implemented.
The first level involves mitigating bottlenecks in fixed facilities, leaving unit operations in place but scheduling or locating them more effectively. The flip side of level one is adopting new technologies to enable faster processing. “You’re not really eliminating any step or operation, so the potential impact on viral safety is minimal,” says Dr. Phillips.
Compressing processes by connecting unit operations, the second level, presents modest challenges regarding viral safety. However, such strategies, he notes, “need not be serious, provided one remains vigilant that virus removal is not compromised.”
The third level of process intensification—continuous processing—is where issues raised by connecting unit operations appear “in spades,” according to Dr. Phillips. “Continuous processing is a huge change, a revolutionary development with profound implications for viral safety.”
As with some revolutions, however, this one will be slow in coming. Although many companies and suppliers are evaluating continuous processing, Dr. Phillips cautions not to expect the coup to occur overnight. “There are regulatory concerns and technical gaps in the ability to implement continuous processing in the near future,” he explains. For example, current-generation sensors to ensure reliable operation of continuous processes are lacking, as are control strategies. Dr. Phillips believes that as the technology improves and regulations coalesce around continuous processing, viral safety within that environment will catch up. “But don’t expect it for at least five to ten years.”
A more modest implementation of continuous processing exists within the confines of individual unit operations. Perfusion cell culture and variants of simulated moving bed chromatography are two examples. Another is inline low pH virus inactivation. The discovery that low-pH incubation could be significantly shorter than the usual one-hour hold, coupled with the inconvenience of standard two-tank virus inactivation, led Dr. Phillips’ group to investigate continuous low-pH treatment of process fluid as it emerged from a protein A chromatography capture column, obviating the need for an intermediate incubation/hold step.
When viewed against the backdrop of an entire process, these individual steps less represent continuous processing than optimized or streamlined batch operations in which feedstock enters, is processed, and then awaits the next step. Under ideal continuous operation, process fluids feed directly and continuously into and through operations, and purified product continuously flows out. Continuous or “next-generation” bioprocessing promises huge advances in productivity, but process developers must be aware of how those advances could affect viral safety. The improvements as one progresses along various levels of process intensification also bear a potential cost.
Herb Lutz, global principal consultant at Millipore- Sigma, described recent results where a tangential flow filtration-based protein concentration step was performed before flow-through anion-exchange chromatography polishing. “We reduced the volume of the protein solution by a factor of four to make the chromatography column more efficient, but we were obliged to test how this might affect viral clearance,” said Lutz. Using this highly concentrated feed, they demonstrated consistent 5 logs clearance of MVM and XMuLV at high product loadings, confirming that viral clearance was maintained in the smaller footprint process.
“We’re running processes in new ways,” he says. “The data doesn’t yet exist to guide processors on the implications of all aspects of how process changes impact viral clearance.
Conventional viral-clearance testing assumes that purification steps behave uniformly throughout their operation. Aliquots of feed are spiked with model virus, subjected to normal filtration or chromatography, and the concentration of virus before and after the operation are measured and results are reported as a log reduction. Lutz, however, does not believe this accurately represents conditions of continuous processing:
“Standard virus-spiking strategies are inadequate when the feed solution changes because a protein peak is coming through or some other event is occurring,” he maintains.
To better assess such operations, Lutz developed a technique termed inline spiking, which enables monitoring of viral clearance under more representative conditions, when the process feed changes due to fluctuating concentrations of proteins and salts.
It turns out that in most cases (e.g., during cationexchange chromatography), virus retention is fairly constant over a wide range of protein concentrations. Nevertheless, the value of inline spiking is that for a minor investment in time it provides a clear answer. “Regulators like that,” Lutz adds.
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Appears with permission from copyright Genetic Engineering & Biotechnology News