By Cynthia Challener, PhD
The introduction of adventitious viral agents is a recognized, inherent risk of biologic drug production that is well addressed by international regulatory requirements for analysis of upstream inputs and viral clearance during downstream processing. The success of viral clearance depends on the selection of appropriate methods that ensure the removal or destruction of any viruses without affecting the target protein. Several factors must be considered when making this selection: the clearance requirements, the properties of the target protein, the mechanism(s) of the clearance method(s), and the impact of process parameters.
Many methods to choose from
There are numerous methods that can be employed to achieve viral clearance during downstream biopharmaceutical processing, some that are better understood than others. “Viral clearance steps can be broadly classified into three basic categories: well-understood steps that are known to consistently provide robust virus reduction; lesser understood steps that have been somewhat characterized but provide lower or variable virus reductions; and steps that are not characterized, are not particularly effective, or are unpredictable,” says Daniel Strauss, a senior scientist with Asahi Kasei Bioprocess America.
Ideally, methods from the first category, which include inactivation mechanisms such as low pH, detergent, or temperature hold steps and robust removal steps, such as virus filtration that clear a broad range of potential contaminants, are used. “These dedicated viral clearance steps are well established in the industry, and as long as they are operated within established ranges and the product quality is not impacted, virus reduction can usually be assured,” Strauss notes.
Processes from the other categories are typically steps optimized for product purification and not dedicated for viral clearance. These steps can, according to Strauss, provide acceptable clearance but they often require more effort to optimize and validate while providing lower log reduction values (LRVs). Literature data can help in evaluating and planning effective optimization of these steps.
A matter of balance
Implementation of viral clearance steps must be accomplished without affecting the integrity of the protein product, which can be a challenge because physiochemical methods can induce the aggregation, fragmentation, or other undesired effects on some molecules. It is essential to have an understanding of the protein and its characteristics, according to Kathy Remington, a principal scientist in BioReliance’s Development Services group. “Knowing the protein’s tolerance for low pH, detergent, heat, etc., will help to direct the selection of a viral inactivation step. Understanding the size of the protein and its filterability is also necessary for selecting an appropriate virus reduction filter,” she explains.
Nanofiltration is added to most processes (except for viral gene therapies) and provides excellent clearance of small (parvovirus) to large viruses (e.g., murine leukemia virus [MuLV]), according to Frank Riske, a senior consultant with BioProcess Technology Consultants and previously a senior director with Genzyme. “Nanofiltration has simplified the method selection process because it is almost always included unless the product molecule is too large to pass through a 20-nm pore. It is a gentle, effective method that works universally on small, large, enveloped, non-enveloped DNA and RNA viruses,” he observes.
A second inactivation/removal step is also frequently included, and the specific type is determined by the tolerance of the target protein for low pH (~3–4), solvent (tri-(n-butyl) phosphate;TNBP)/detergent, etc. In some cases, Riske notes that this inactivation step is incorporated into a chromatography method, such as in a solvent wash. “Column chromatography will frequently be effective for virus reduction, but the degree of reduction is dependent on the resin type (mode of action), the conditions used for the separation, and the characteristics of the target. Typically, anion exchangers in either a flow-through or bind/elute mode are effective for separating viruses from biologic products,” he remarks.
The importance of early data development
Ideally, according to Remington, the development of a viral clearance strategy should be done in conjunction with development of the protein purification strategy using actual viral reduction data. Because viral clearance studies are typically outsourced, particularly by smaller companies, these data are often not generated until they are needed to support a regulatory submission. “One common pitfall is to make decisions about viral clearance methods at early stages of development without fully considering the implications if all goes well and the molecule advances to late-stage development and commercial production,” agrees Strauss.
For well-understood steps, such as detergent or low pH inactivation steps, Remington notes that this approach is usually successful, but for others, and particularly chromatography steps, the lack of development clearance data may prove problematic. Riske adds that these problems generally arise because the column and purification conditions are chosen based on the ability to produce a purified protein that meets specifications and are developed to maximize purity with reasonable recovery. Only then is a chromatography step tested for viral clearance. “One solution is to add a step specifically to reduce viruses flow-through anion exchange may work—or the separation conditions can be modified,” Riske says.
“It must be understood that viruses are complex proteins that are impacted by pH, conductivity, and other operating parameters, just as is the protein product. Determination of the mechanism of virus removal/inactivation and understanding the impact of process parameters on viral removal/inactivation is the best way to ensure good clearance, but the development of viral clearance studies are often required to obtain this information,” Remington says. She adds that mapping the design space of a process step for viral clearance is the best way to optimize the step for virus reduction and is the approach generally used to understand the clearance potential of a step that will be included in a manufacturing platform.
Lot-to-lot-variation in both product feedstocks and consumables is another factor that is often not considered during development because it does not generally have an impact when preparing a small number of batches to supply clinical trials. “When those processes advance to commercial production with regular manufacturing and carefully orchestrated facility constraints, however, any variation in performance from batch to batch can disrupt production timelines,” asserts Strauss. The selection of robust, highly scalable methods that are not affected by variations in feedstocks and consumables is essential for achieving reliable and consistent processes in terms of both their viral clearance capability and performance in the plant is crucial for avoiding these issues.
A need for defined clearance targets
Having a broad perspective on various regulatory requirements and defined clearance targets is also important when selecting viral clearance methods. Regulatory requirements vary by country, individual agency, the phase of development, and the contamination risk profile for a particular product, according to Strauss. Target clearance values also depend on whether certain viruses are known to be present in the host organism or if the process is intended to clear a broad range of virus types. “An inadequate understanding of the clearance requirements can result in failure to hit the needed clearance targets and the need for additional validation studies or viral clearance steps. Alternatively, resources may be wasted achieving excessive removal values,” he says.
Improvements in filtration
Advances in filtration technology are having the biggest impact on viral clearance, according to industry experts. The newer generation virus filters have higher fluxes, larger product capacities, and increased virus removal capabilities, with some also offering steam-in-place (SIP) capabilities, which enable their use in closed processes. Others do not require prefiltration to avoid filter clogging. “These advances allow for implementation of high capacity, cost-effective virus filtration steps that easily achieve required virus reduction results,” Strauss states.
New chromatography membranes also simplify the viral clearance assessment, according to Remington. “These disposable membranes remove the need for evaluation of sanitization steps and the need to evaluate aged resins. In addition, the risk of a potential viral contaminant being carried over from run to run is eliminated with disposable technologies,” she says. The increased availability and understanding of membrane adsorbers as viral clearance tools, such as anion exchange (AEX) membranes, allow these technologies to serve as good backup solutions for virus removal that can be dropped into many processes with minimal development work and without many of the concerns of adding an additional chromatography step, according to Strauss. Configurations of older methods, such ultraviolet-C (UV-C) high-temperature short-time (HTST) inactivation are also facilitating the implementation of these older methods into current downstream biopharmaceutical processes, according to Remington.
Holistic approach
Unfortunately, there is no one-size-fits-all method that provides complete clearance of all viruses in all processes. The industry is, however, developing a much better understanding of the mechanisms of virus reduction by many of the commonly-used methods. Remington believes that this increased understanding will facilitate the design of processes that optimize viral safety. Taking a holistic approach is the most effective means of achieving viral control, according to Riske. “While traditionally viral clearance has been achieved during the downstream processing of biopharmaceuticals, increasingly there is a focus on ensuring that raw materials are free of adventitious agents. This holistic approach tackles potential contamination by adventitious agents throughout the entire process.”
Filter sterilization of production media using nanofilters is one possible measure that companies can take, but Riske notes that nanofilter prices are currently too high and their throughput too low for practical use in upstream processes at large scale. Filter manufacturers are working to address these issues, and Riske looks forward to seeing solutions on the market in the future. Another important development for the biopharmaceutical industry has been the shift in perspective of viral clearance.
“For many years, the industry approached viral clearance as an exercise in validation and not something that needed to be developed, optimized, and fully understood at a mechanistic level. Recently, however, there have been big improvements in available data with many more articles in peer-reviewed journals and an increase in published conference proceedings. These data have helped significantly to improve method selection, development, and validation, and they have also influenced the stances of regulatory agencies in ways that have eased the regulatory process across the industry,” asserts Strauss. He hopes that companies will continue to be forthcoming with their viral clearance knowledge and experiences as a means to demonstrate to the agencies and to the public the industry’s commitment to patient safety.
About the Author
Cynthia A. Challener, PhD is a contributing editor to BioPharm International.