Viral clearance processes and guidance must evolve along with newer biotherapeutic modalities.
By Feliza Mirasol
Ensuring viral clearance (i.e., removal) in downstream purification has become increasingly challenging based upon the increasing range of product classes emerging. Because viruses are typically smaller than downstream impurities such as host cell proteins (HCPs) and other extracellular debris, they require meticulous filtration, even in more established processes such as monoclonal antibody production.
However, demonstrating viral clearance is even more difficult with the newer biotherapeutic modalities being developed today such as viral vector-based gene therapies. Many of these new and evolving biotherapeutics use viruses or virus-related approaches in their manufacturing processes.
Clearance challenges
The challenge of demonstrating viral clearance in downstream bioprocessing depends on multiple factors. For most traditional biological products, viral removal is theoretical—in part, because infectious virus is not usually present in the production process, says James Berrie, PhD, technical director, Lonza. There are exceptions, however, such as gene therapies, for which manufacturing processes are virus-based (e.g., in viral vector-based processes). In these cases, many of the viral removal or inactivation technologies that are typically applied to “traditional” biologics will not work.
Generally, viral clearance methods for these processes remove known impurities, such as DNA and HCP, by using assays that were developed and evaluated for a specific cell line, says Berrie. Thus, for example, one reduces the HCPs generated by a cell line by manipulating the buffers and chromatography chemistries to separate the product. In such cases, viruses are absent, and their presence would represent a contamination of the process, Berrie says.
For traditional downstream processing templates, such as monoclonal antibody (mAb) purification, the technologies used to ensure virus removal are well established and their effectiveness has been confirmed by the safety record of the therapeutics they were used to manufacture, adds Darren Verlenden, head of BioProcessing at MilliporeSigma.
Potential viral separation challenges center around the diversity or variety of potential viral contaminants, he says. First, dependent on the production cell line, there may be a number of viruses with different physicochemical characteristics to consider, Verlenden explains. Secondly, the quantity of virus or virus-like particles in these different cell lines may potentially be quite high, he says, requiring multiple efficient virus-removal steps to deliver the clearance required for safety.
Finally, there is the issue of parvoviruses, which are only slightly bigger than the molecules being produced, Verlenden says. In addition, parvoviruses are highly resistant to physicochemical treatments, and manufacturers typically use filtration to remove them.“Although [these methods] are highly effective, the challenge is optimizing the balance between high levels of virus retention on the filter and high recovery levels for the molecule of interest downstream of the filter,” he states.
However, manipulating one process technology to remove a specific virus may not be appropriate for other virus types, Berrie explains.“In small-scale viral clearance studies, these differences are addressed by spiking a panel of viruses with a range of physicochemical parameters to challenge the purification process,” he says.For example, enveloped vs. non-enveloped viruses may be used to testinactivation technologies, or small vs. large viruses such as parvovirus, to test virus-retentive filters. In the latter case, size-based separation may be challenged under conditions of interrupted flow during manufacturing operations, Berrie states.
Prevent, detect, remove: a holistic approach
As Verlenden points out, product safety relies on following the basic tenet of “Prevent, Detect, and Remove,” a holistic approach that requires:
• Careful selection and pre-treatment of raw materials to prevent viruses from entering upstream processes
• Testing for the presence of viruses
• Implementing appropriate purification and filtration technologies to remove viruses downstream.
“As we look at new technologies for improving the efficiency of molecule separation, we always evaluate them based on their ability to remove a virus,” Verlenden says, noting that most downstream templates use a combination of chromatographic separations, inactivation technologies, and filtration to accomplish this.
Despite technology advances, “optimized” may not be the best term to use for viral clearance, says Berrie. After all, he reasons, how can you say you’ve removed something from a process when its presence may be unknown, and its nature unpredictable?
In addition, there is great variability between viruses. “A process parameter optimized for removing one type of virus will not be optimal for all virus types. As with any molecule class, removal will depend on specific parameters (e.g., isolectric point [pI], hydrodynamic radius, and surface charge, under certain process conditions),” he says.
This variability has changed the way that many companies are approaching viral clearance. At Lonza, Berrie explains, automation is being used in virus reduction filtrations (VRFs) to improve the assurance that viral clearance claims have been attained.
This is accomplished by controlling liquid flow during a VRF step based upon the pressure differential across the VRF filter itself, says Berrie. So far, he adds, this approach has improved overall pressure control and reduced perturbations, compared with processes where human intervention is required to control flow based upon pressure alarm limits.
In downstream processing, chromatographic separations leverage interactions through affinity, charge, or hydrophobicity, says Verlenden. Improvements in chromatographic separations designed to maximize HCP and impurity removal have often resulted in higher levels of viral clearance as well, he says, pointing to MilliporeSigma’s chromatography resin platform, Eshmuno, as an example of a product that offers improved separation of molecules from impurities, while also providing some level of viral clearance.
Detergents and membrane clearance
Viral inactivation using low pH or detergents delivers high levels of enveloped viral clearance under standard conditions, Verlenden says, as exemplified by the recent release of standard conditions from the American Society for Testing and Measurement (ASTM) (1). Although environmental concerns will limit the use of traditional detergents in the near future, Verlenden says, MilliporeSigma is developing a detergent that minimizes environmental impact while providing strong viral inactivation levels.
The virus filter remains the most reliable technology for removing viruses (both enveloped and non-enveloped types) in downstream bioprocessing, Verlenden notes. Improvements in his company’s Viresolve Pro product enable viral clearance effectively in both traditional biologics manufacturing as well as in evolving bioprocesses.
Ultimately, the effectiveness of VRF depends on flow, says Berrie. “If viruses are similar in size to the pore size of the VRF membrane, the extent to which a filter can reduce virus titer depends on the flow conditions applied to the VRF. Interrupting flow can exacerbate the passage of small viruses into the product filtrate,” he says.
As with most phenomena, however, there are always rare exceptions in which no breakthrough of small viruses can be demonstrated, Berrie continues. Such exceptions depend on other conditions, such as feedstream pH or conductivity. “The vital principle is that the small-scale model used for the viral clearance study must represent the manufacturing-scale operation,” he says, “and ideally it should represent a ‘worst case’ scenario compared with the large-scale operation,” he says.
During development, it is important to consider the pressure differential applied within the design space, Berrie says, based upon the concept of small virus passage and both upper and lower pressure limits. In addition, he notes, the number and duration of permitted pressure interruptions should be defined for a given process and built into the strategy for the small-scale evaluation. In the end, only the virus reduction filter itself can be claimed as a virus removal filter, because other filters in the process are not evaluated for their ability to reduce virus titer, Berrie adds.
Process intensification is having an impact on downstream virus removal. “Many biomanufacturers are moving towards more intensified processing paradigms, in which the molecule concentrations are higher than they are in more traditional processes, and there may be some connected unit operations,” says Verlenden, noting that this trend challenges suppliers to ensure that filters can handle operating at lower flux for longer periods of time without losing efficiency or their ability to retain viruses. “In addition, processing may be temporarily paused during runs, and it is critically important that these temporary depressurization events do not compromise viral safety,” he says.
Next-gen biopharma processing
Recent innovations, particularly in filter membranes and chromatography resins, have further helped to improve viral clearance performance.
“In general, chromatography resins are developed for their ability to remove actual or known process impurities rather than viruses. However, viruses, like other impurities, have characteristics such as pI, which may be leveraged for the development of their removal.Resin and membrane manufacturers always provide data on virus reduction, as this is always anticipated as a consideration in purification process development,” says Berrie.
New product innovations and the establishment of new operating conditions for legacy products have been instrumental to meeting evolving processing needs, Verlenden adds. As an example, he points to recent studies conducted on MilliporeSigma’s single-use chromatography membrane, Natrix Q. Results showed that this chromatography membrane can work across a broad range of operating conditions, offering not only efficient removal of impurities but excellence clearance of both enveloped and non-enveloped viruses, Verlenden says. The product was designed for manufacturers who seek to maximize the flexibility and efficiencies of single-use manufacturing, he adds.
In terms of traditional viral clearance products, Verlenden continues, studies have shown that concentrating protein solutions using single pass tangential flow filtration (SPTFF) before loading them onto a resin column, such as Eshmuno Q resin column (MilliporeSigma), improves the productivity of the chromatography operation while maintaining efficient viral clearance.
Another area of intense product development is in systems that will support intensified and continuous in-line viral inactivation, Verlenden says. “As we look to develop more rapid manufacturing processes technologies around chromatography intensification, such as continuous chromatography, differing approaches to scaled-down models are required. Developing new scaled-down models presents a challenge, especially in the context of ever-shortening timelines,” adds Berrie.
However, developers are evolving new approaches to viral clearance that fit into the shorter development times that have become the norm. For instance, Lonza has a short timeline offering (of 12 months) for filing an investigational new drug (IND) application, which includes a virus-reduction study. Generally, the samples for a viral clearance study are taken from a manufacturing-scale run, which places the data generation late in the overall timeline, says Berrie.
This can require a significant length of time for virus assays to be completed, and for these assays to be evaluated prior to the spiking runs, he explains. However, Lonza has come up with new ways to meet timeline challenges while delivering the required viral clearance within a year. These approaches involve, in part, looking at earlier points in the timeline from which to begin viral clearance evaluation. In addition, pre-study testing efficiencies are leveraged from the timeline based upon a platform approach. “Multiple efficiencies combine to bring in the data to allow customers to meet a relatively aggressive timeline,” Berrie says.
Significant challenges to viral clearance are being encountered with cell and gene therapies, however. For instance, some specific viral clearance steps may not be suitable because the product of interest may either be inactivated or removed, Berrie clarifies.
Guidance is needed
Compounding all these challenges is the fact that published regulatory guidances have been based on traditional biologics, such as mAbs and related Protein A binders, says Berrie, noting a recent BioPhorum Development Group (BPDG) survey. Many companies and biopharma professionals, the survey found, would welcome more regulatory guidance on what level of viral clearance capability must be demonstrated in the newer types of biological therapeutics, he says.
More clarity and specific guidance on viral clearance considerations is expected in a revised viral safety being developed by the International Council for Harmonization (ICH) as part of its Q5A guideline (ICH Q5A), Berrie says (2). This is expected within the next few years, he adds.
Like developers and other contract development and manufacturing organizatons (CDMOs), Lonza must deal with increasing numbers of complex protein processes that may not lend themselves to a platform-based approach. These processes are subject to unknown or unpredictable capacity for viral clearance. As a result, the trend today is toward shorter viral clearance study timelines, performed either earlier in the timeline or as part of developmental viral clearance evaluations, Berrie says.
Where the choice of viral clearance approach for mAbs is clear cut and well understood, says Verlenden, even in this area, challenges are becoming visible as modalities get more complex. “The industry has a way to go in developing a range of viral clearance products that enable efficient separation of more complex mAbs, such as bispecific or Fc-fusion proteins,” he says.
Another challenge will be developing viral clearance approaches that synch with continuous manufacturing.Technology developers are actively focused on coming up with systems that will enable all unit operations, including low pH viral inactivation, to run continuously, he says.
In the end, the greatest challenge for manufacturers, CDMOs, and vendors alike is the sheer diversity of biopharmaceuticals being developed throughout the world today. It’s “awe-inspiring,” Berrie says. For viral clearance technology, the difficulties ahead will lie in areas such as viral gene therapies, in which viruses are not only the products but must also be separated from helper viruses or potentially adventitious contaminants, in order to assure product and patient safety, he adds.
As more of these new therapies are scaled up, technology vendors continue to fine tune downstream filtration and separation technologies. Among these crucial areas is viral clearance. Equipment and methods are changing to help biomanufacturers meet their goals in both traditional and new therapeutic areas.
References
1. ASTM, “Proposed Standard for Inactivation of Enveloped Viruses Being Developed by ASTM Pharmaceutical Committee,” Press Release, March 4, 2013.
2. ICH, Q5A Quality of Biotechnological Products: Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human or Animal Origin (1999).