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New biologic modalities, such as cell and gene therapies, pose increasing difficulties in viral clearance testing methods.
Testing for viral clearance in downstream bioprocessing is growing increasingly challenging for new biologic modalities as the processes to make these therapies are themselves becoming more complex. For example, gene therapies rely on the use of viral vectors in the manufacturing process, so distinguishing viral vector components from virus contamination requires sensitive and discriminating testing methods. Do current techniques address these challenges or is further innovation required to ensure continued product quality and purity?
There are two primary factors that can increase the difficulty of viral clearance, says Alex Schwartz, product specialist, Viral Clearance, Sartorius. First, the raw material inputs for a number of these new biologic modalities require the use of high-risk items, such as animal- or human-derived sera, he explains. The use of these high-risk materials means there is a significant increase in viral contamination risk because these materials are frequently contaminated, and testing is not always 100% effective at screening, he cautions.
Secondly, these new modalities have significantly reduced downstream unit operations. “While viral vectors cannot be processed through traditional viral inactivation methods, they often only rely on one or two column processes that may or may not provide robust viral clearance greater than four log reduction values (LRV),” Schwartz states. Thus, in cell therapies, there are no effective chromatographic techniques, and, like the viral vector space, no viral inactivation is considered safe for the product. Additional efforts are thus usually taken as a viral barrier in the cell culture processing, he adds.
Marc Breton, group leader III, Viral Clearance Services, Eurofins BioPharma Product Testing, adds that, for these new modalities, the challenge to overcome is to accurately distinguish native (product related) virus from the spiking virus used to challenge a process step.
Meanwhile, Joshua Huffer, technical specialist, Field Technology Management, MilliporeSigma, emphasizes that the most difficult aspect of many of these advanced medicinal therapeutic products (ATMPs) is the fact that it is difficult to find a processing step that will selectively inactivate or remove the potential viral contaminants without inactivating the biological product. “For example, retroviral or lentiviral gene therapy vectors will be inactivated by steps that inactivate enveloped viruses. These products are relatively large, so that virus filtration cannot be used,” he points out.
The same difficulties also exist for cell therapies. Apart from non-enveloped viral therapies (e.g., adeno-associated virus [AAV] viral vectors), viral clearance is not an option for cell and gene therapies, Huffer says. “In the absence of any potential viral clearance, extra scrutiny is placed on the cell line characterization and testing the input raw materials to reduce the risk of any potential viral contamination,” he explains. “For enveloped viral vectors and cell therapies, we recommend the use of barrier methods to treat the cell culture medium and medium components.”
Methods, such as high-temperature, short-time (HTST) and virus reduction filtration, reduce the risk of the potential entry of a viral contaminant into manufacturing, Huffer adds, explaining that barrier filters are designed for this purpose. “This mitigation method is well-suited to the smaller batch sizes used in cell therapies and some gene therapy products,” Huffer states.
Meanwhile, non-enveloped viruses, such as AAV, can follow more traditional viral clearance routes. “These viruses are much more resistant to detergent and low pH treatment steps, which will inactivate enveloped viruses. The use of viral filters with larger pore sizes (e.g., 35 nm, 50 nm, 70 nm) will allow the small AAV vector to pass through, while retaining larger enveloped viruses. An inactivation step and a viral filtration step will add robust clearance of enveloped viruses to the manufacturing process for an AAV product. Affinity chromatography and anion exchange chromatography can potentially contribute to viral reduction from the purification stream,” Huffer explains.
With these challenges, having the right approach and techniques is important for successful virus removal to ensure product quality/purity for ATMPs. Although the number of chromatography steps for a gene or cell therapy are limited compared to other fields, such as monoclonal antibody (mAb) products, there are still common steps that can be used for most gene therapy studies, including affinity and anion exchange chromatography, says Tareq Z. Jaber, PhD, manager, Process Evaluation, Charles River Laboratories. Jaber explains that these steps can cover both enveloped and non-enveloped viruses, including parvovirus models (in AAV products) or retrovirus models (in lentiviral products). “Including other steps, such as detergent inactivation, will depend on the product nature itself. If AAV is used, detergent can be applied since AAV shows resistance to detergent inactivation while retrovirus or herpes virus vector products will be sensitive to detergent,” he states.
Polymerase chain reaction (PCR) techniques are useful for titering virus concentrations in these complex systems—especially for gene therapy products, which have potential native virus that could interfere or even obscure the result in an infectivity assay, adds Breton. “Due to their specificity, PCR techniques can solve this challenge, but are unable to distinguish between active and inactive virus. To overcome this challenge, analyzing virus titers determined by both PCR and infectivity techniques can be helpful, depending on the process step being challenged,” he says.
For an AAV vector, an enveloped inactivation step and virus filtration using a large pore filter will usually provide effective enveloped viral clearance, Huffer says. “For cell and enveloped vector gene therapy products, viral clearance steps are not feasible and, therefore, selecting and testing the cell lines and raw materials used in the manufacturing process are key to reducing the risk of potential viral contamination. The use of a barrier method to prevent entry of a viral contaminant into the process stream should also be a key consideration,” he states.
Timeline constraints are another challenge that tends to be a common concern for viral clearance testing, regardless of product type, Breton says. “I always advise that early communication with your viral clearance vendor is key to the quality and performance efficiency of any viral clearance study—this is doubly important with novel techniques and products,” he says.
“Time is always a concern, especially these days,” Jaber agrees. He points out, however, that for clinical Phase I/II-stage programs, regulatory agencies do not require viral clearance for all gene therapy products studies, although the agencies may recommend it. “If there is no risk of copurification of a helper virus or of an adventitious virus (e.g., from the cell line, such as an endogenous retrovirus or rhabdovirus, or from raw material) agencies do not require viral clearance studies. However, it is regarded mandatory to analyze for Phase III studies and commercial release,” he states.
If the downstream process has clearance potential (chromatography steps, for example), the clearance should be analyzed to demonstrate safety of the product, Jaber adds. “When required (in case of a known contaminant or copurifying virus), viral clearance should be adequately demonstrated as per the recommendations in ICH [International Council for Harmonisation] Q5A (1),” he says.
Huffer adds that for time-sensitive therapies, such as autologous cell therapies, viral clearance steps are not an option. “Rapid detection of potential contaminants—viral and microbial—is critical for these time-sensitive therapies. Making use of rapid testing options, such as rapid sterility, quantitative polymerase chain reaction (qPCR), or other molecular methods for detection of mycoplasma and other known contaminants, are valuable techniques to ensure patient safety,” he states.
Schwartz adds that, since viral clearance should still be viewed as a platform approach to cell therapies, most viral clearance claims should not impact patient material or their specific timing needs. “Outside of viral clearance, what can make an impact is the speed of QC [quality control] virology testing required to return patient cells to the clinic,” he says.
The need to improve viral clearance for these new biologic modalities requires innovation in thinking as well as in technology. Inactivation and filtration steps used for enveloped viral clearance in AAV manufacturing processes are not new but can still provide robust viral clearance of potential enveloped viral contaminants, says Huffer. He also points out that barrier filters used for mitigation of viral contaminants are newer than standard viral reduction filters; however, they are especially designed to reduce the risk of a viral contamination event. “These are valuable in risk mitigation for gene and cell therapy products,” he says.
Huffer adds that new molecular methods, such as next generation sequencing (NGS), can be used to screen process materials going into a manufacturing process that do not have the option of viral clearance. “These new technologies reduce the risk of introducing a viral contaminant into a manufacturing process with limited or no opportunity for viral clearance,” he notes.
NGS can be a significant advantage with the possibilities to detect both known and unknown adventitious viruses, Jaber says, noting that characterized virus stocks and standard process steps will help validate the NGS platforms.
“Cell and gene therapy product manufacturing involves multiple steps, long culture, and processing times, and therefore may allow amplification of adventitious agents. Starting material and raw material qualification and testing [are] critical to ensure the safe manufacture of products. For example: implementing methods to remove or inactivate virus in media or components, development of media that do not require animal- or human-derived raw materials, and use of closed system to avoid contamination by environmental sources,” Jaber includes.
Meanwhile, traditional resin chromatography has been the mainstay of downstream processing for most biologics, Breton says, but with innovations in membrane chromatography, the industry has seen greater interest in these techniques. “For gene therapy products, we are starting to see monolith technologies utilized due to their defined flow path diameters, which are uniquely suited for larger products such as AAVs,” he states.
“Additional chromatographic techniques have been helpful in providing additional product purity and viral safety in the viral vector space,” says Schwartz. “For cell therapies, there are several advances that aid in setting an upstream viral barrier. These include virus retentive filters designed for media and the implementation of closed single use technologies,” he adds.
In discussing the unmet needs in the industry regarding viral clearance studies for cell and gene therapies, Schwartz asserts that there needs to be additional effort around the safety of cell therapies in particular. “Most risk analyses provide only barrier technologies and not true in-process viral clearance options. Having additional downstream tools that provide this clearance without impacting cell viability would change cell therapy processing,” he says. He further states that additional effort can also be made to develop faster and more effective viral clearance testing services to lower the bar for these new modalities to enter the clinic.
“Cell therapies do not typically have any downstream purification that could have the potential to remove or inactivate viruses,” Jaber also adds. Thus, viral clearance is not an applicable test method for these therapies, he states. “Therefore, animal- or human-derived raw materials/ancillary materials used in the production of cell therapy products should be critically reviewed, and if viral clearance is applicable in the production of such materials it should be applied,” he cautions.
Because it is currently difficult to remove viruses from cell or enveloped gene therapies without potentially damaging the product, finding a solution would greatly provide an unmet need for these products, says Huffer. Purification steps that provide an opportunity to selectively inactivate or remove a viral contaminant yet keep the biological product intact and activewould increase viral clearance opportunities for these products. “In the absence of new viral clearance steps for cell and enveloped vector gene therapies, new, sensitive molecular techniques that can identify viral contaminants in starting materials would add to increased viral safety,” he states.
Meanwhile, Breton points out that strong regulatory expectations for new biologic modalities in terms of viral clearance studies is the proverbial “elephant in the room” and that new science and new modalities are rapidly outpacing regulatory determinations. “This leads to feelings of uncertainty but, moreover, can potentially lead to inefficiency in bringing a much-needed product to market,” he states.
1. ICH, Q5A Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human or Animal Origin, Step 4 version (1999).
Feliza Mirasol is the science editor for BioPharm International.
Vol. 34, No. 7
When referring to this article, please cite it as F. Mirasol, “Viral Clearance Testing Increasingly Challenged by New Biologic Modalities,” BioPharm International 34 (7) 44–48 (2021).