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The design of viral clearance studies must keep pace with the quickly evolving biologic drugs industry.
Viral clearance (VC) studies are mandatory for biologic drugs, which are pharmaceutically derived from human and/or animal sources such as cell line-derived recombinant proteins, blood products, vaccines, and critical medical devices. In these studies, the potential of removal and/or inactivation of adventitious and endogenous viruses of certain steps of the manufacturing purification process is addressed in a small-scale approach. Though regulatory guidelines are in place, the design of such a VC study is often not as straightforward as expected. The current guidelines do not keep up with the fast pace of the industry in terms of acquired knowledge, evolvement of new product categories, new methodology, and new production modes, which often leaves room for interpretation. In addition, there are other challenges and pitfalls that need to be taken into consideration when performing VC studies. This article will cover VC studies, their challenges, and the quickly evolving biologic drugs industry.
The first recommendation addressing the VC for biologics produced in cell lines was published by FDA in 1993 (1). Followed by the first VC guideline from the European Medicines Agency (EMA) in 1995, which covers all the categories of medical biological products for human use, with the exception of live viral vaccines, including genetically engineered live vectors (2). After almost 30 years, the reference guidelines do not cover the non cell line-derived biologics, which are also not addressed in raw-material-specific guidelines, including the urine-derived medical products (3) or the human blood plasma products (4). The reference guideline cell line for cell line-derived biologics from the International Council for Harmonisation (ICH), the ICH Q5A (5), was initially released in 1997, revised two years later, and is currently undergoing a long overdue revision. Meanwhile, several other guidelines and recommendations have been released. Despite these documents being in place, for manufacturers to decide on a VC study scope, they still must consider many aspects as guidelines are often only valid in certain countries.
In general, the regulatory guidelines ask biomanufacturers to prove the potential of at least two separate process steps independently. As a consequence, a combination of solvent/detergent and/or low pH treatment followed by a chromatography and virus-retentive filtration is being addressed. When it comes to the virus panel to be tested, the guidelines highly recommend addressing the known or suspected contaminates while covering all biophysical properties in terms of RNA or DNA, enveloped and/or non-enveloped viruses. Therefore, each VC study must be reviewed on a case-by-case basis to evaluate the risk factors associated with each tested product, including raw materials.
In the past 20 years, VC knowledge and data has accumulated especially for Chinese hamster ovary (CHO)-derived recombinant products. Additionally, considerations to reduce the VC study scope (e.g., standardized protocol ASTM E2888-12R19) for low pH treatment has been taken into account in which, under certain requirements, a 5 log10 clearance for murine leukemia virus (MuLV) can be claimed. However, the increase in knowledge and recommendations are only in place for certain territories, which raises the uncertainty and challenges in the industry regarding VC study scopes.
One challenge in VC studies is the demand of a scaled-down version of the process step. When scaling down is accomplished successfully, and its validity is demonstrated, a second challenge follows in that this established scaled-down version has to be communicated to a VC testing team. This team works in a laboratory setting, which is separated from the production site to prevent contamination of manufacturing processes. Often times, the VC studies are carried out by an external contract research organization. Therefore, a good understanding of the process and efficient communication between the manufacturer and the VC testing team are crucial for a seamless VC study.
Recently, as the industry optimizes their manufacturing processes to increase profit, new challenges have arisen. For instance, product concentrations are massively increasing in manufacturing. This increases the general risk of aggregation, especially upon addition of a high-titer virus spike that is being added for VC testing. In many cases, this also requires additional adjustments of common de-coupled VC study set-ups to inline process. This is a specific process where pre-filtering is employed directly before the chromatography or virus-retentive filter, which, due to the more sensitive material, cannot be de-coupled. Even though data obtained in recent years indicate that some pre-filters do indeed have the potential to remove viruses, the potential cannot be claimed. Authorities argue that integrity tests for these filters are not in place. Hence, the impact of the pre-filter on its own has to be addressed in addition to the inline set-up.
Another aspect is that biomanufacturing is transitioning from batch-mode processing to continuous processing (CP). This transition, of course, bears an exceptional challenge when it comes to establishing a scaled-down model and calls for innovative alternatives, which results in specifically tailored scaled-down models for VC testing purposes that should take into consideration the integration of linked-unit operations. Additional key issues for both increased protein concentrations and CP in manufacturing include the increases in volumetric throughput and product load across a retentive filter, which will cause virus breakthrough once critical thresholds are met. Thus, suitable sizing and scaling of the retentive filter is of paramount importance to accommodate the increased load.
Maintenance of a high standard of purity is a requirement of any biotherapeutic products. Many of the biotherapeutic products utilize cell culture for production, which increases the risk of contamination. Therefore, it is crucial for VC to be part of the known three principles—selection, testing, and reduction—to prevent the transmission of pathogens, in addition to sourcing and testing. Recently, it was shown that no manufacturer is immune to contamination events (6). In this study, though only 18 cases of contamination were reported in 35 years (6), contamination is still a real threat to all industries, including gene therapy. Contamination can occur in all stages of a product lifecycle. According to Barone et al., most of the contamination in CHO-cell-derived products originated from raw materials or intermediate components, while most of the contamination in human/primate cell lines originated from manufacturing operators or the cell line itself (6).
What about gene therapy products? What is the source of contamination? Considering the adeno-associated virus (AAV) system as an example, cell lines and helper viruses can be the potential source of contamination. Therefore, biological starting materials for gene therapy products are considered key in the assessment of viral safety testing and VC. The adventitious agents can be introduced into the manufacturing process using those biological starting materials, reagents, and raw materials such as animal-derived serum. Moreover, over the past decade, there is a steady increase in investigational new drug (IND) filings of gene therapy products. Because of the backlog for manufacturing, which is a result of limited manufacturing facilities, gene therapy products are increasingly manufactured at multi-product facilities. This situation increases the possibility of unintentional introduction of adventitious agents into the manufacturing process due to cross-contamination at multi-product facilities and raises several questions: what is the best course of action for manufactures to employ? Are there any specific guidelines that can be followed to evaluate if VC studies are needed, and how they can be applied? Unfortunately, there are no current and specific guidelines to direct the gene therapy industry to the required steps to follow regarding VC validation. Based on regulatory agencies, especially FDA, the current available documents might be used as reference guidelines. Some of these documents include the ICH Q5A Quality of Biotechnological Products: Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human or Animal Origin and FDA’s guidance, Characterization and Qualification of Cell Substrates and Other Biological Materials Used in the Production of Viral Vaccines for Infectious Disease Indications.
The principles of ICH Q5A cover topics that can be applied to gene therapy products. For instance, according to the ICH Q5A document, contamination could arise from the cell line starting material by use of contaminated cell substrate or by endogenous contaminants, which can be applied to some gene therapy products. Systems that use baby hamster kidney (BHK) cells with potential retrovirus-like particles (RVLPs); systems that use human embryonic kidney 293 (HEK293 cells) with potential adenovirus contamination; and systems that use Spodoptera frugiperda 9 (SF9) cells with potential rhabdovirus contamination are examples of cell-line-derived contamination risks. Other sources of contamination include raw materials serum/medium, process-related components, such as helper viruses (e.g., baculovirus and adenovirus), and manufacturing operators or the facilities themselves.
It must be kept in mind that VC is not required for all early stages of gene therapy products as it is in recombinant protein products. 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), then a VC study might not be required in the early stages in such cases. The risk assessment for adventitious agents should be performed by each sponsor on a case-by-case basis to assess if VC is needed.
On the other hand, VC is required in certain conditions. The baculovirus/SF9 system can be used as an example of how to address some of the challenges that a system faces under a VC evaluation. Although the helper virus is not a human virus, and the system is mainly serum-free, there is a potential of detecting contaminants in the final product. There are two common sources of contaminations: Sf-rhabdovirus (Sf-RhV) and the baculovirus helper virus itself. Sf-RhV was one source of contamination that was reported in Sf9 cells (7). Small amounts of Sf-RhV particles in the final product may be immunogenic and can cause inflammation. Meanwhile, regarding the baculovirus helper virus, residues of this helper virus were detected in the final product, and those residues also have the potential to activate the innate immune system. Therefore, a VC study should be applied in this case.
One of the challenges in VC studies arises when it is applied to Sf9/baculovirus products because high residues of baculovirus are detected in, not only the harvest material and affinity chromatography load, but also the affinity product and anion exchange chromatography load material. These high residues of virus can impact the sensitivity of VC assays (either in a cell-based assay or in a quantitative polymerase chain reaction [PCR]). It is difficult to overcome the high-endogenous-virus-level background without applying significantly high non-toxic/non-interfering dilutions, which, consequently, will impact the titration limit of detection. This background might be higher than the total load titer of the baculovirus model that is spiked in the load material during the performance of the VC study.
One of the options to overcoming this issue is to use the residues of baculovirus in the tested material as the target virus to be removed or inactivated by the evaluated steps in the VC study, instead of spiking an additional baculovirus in the material.However, the obstacle here is that all the titration assay systems that are used by VC vendors are validated based on their own cell banks and virus banks. Each SF9/baculovirus system’s manufacturer must first establish a validation study at the VC facility to validate the titration assay for their specific baculovirus on the vendor’s cell system. This validated assay can be used mainly in the detergent treatment and affinity chromatography steps in VC studies.
What about other gene therapy viral vector systems, such as lentivirus and herpes simplex virus (HSV)? While it is still applicable to evaluate inactivation steps (e.g., detergent and heat treatments, and a nano-filtration step with certain sizes, such as 30–35-nm filters) in VC studies for AAV systems, these steps cannot be considered with gene therapy products using enveloped virus systems that are larger sizes than AAV. Chromatography steps might be the only options that can be employed to evaluate the downstream processes.
Because the possibilities of applying virus clearance steps during production are limited for many types of gene therapy products, the viral safety of these products should be ensured with the application of a combination of measures, including selection and control of starting materials, raw materials, and equipment. One possible solution involves the prevention of contamination at the upstream level, such as, for instance, implementing methods to remove or inactivate viruses in media or components (e.g., ultraviolet-C, irradiation, high temperature, nanofiltration), development of media that do not require animal/human-derived raw materials, and use of closed systems to avoid contamination by environmental sources, where applicable.
The most common downstream steps in gene therapy products that can be evaluated for VC studies are summarized in Table I.
Finally, the viral safety of each gene therapy must be ensured; this procedure is summarized in EMA’s Guideline on the Quality, Non-clinical and Clinical Aspects of Gene Therapy Medicinal Products:
“Contamination with extraneous viruses and residues of viruses used during production, such as production and helper viruses needs to be excluded as far as possible … Rigorous testing of seed and cell banks, intermediates, and end products for the presence of adventitious virus needs to be conducted in accordance with principles outlined in ICH guideline Q5A (R1)” (8).
The guideline highlighted the importance of testing raw materials of biological origin thoroughly or manufacturing them by production processes that can remove/inactivate adventitious and endogenous viruses. Therefore, when applicable, VC studies should be considered to validate these production processes to determine the reduction factors for each of the tested steps (8).
1. FDA, Points to Consider in the Characterization of Cell Lines Used to Produce Biologics (CBER, 1993).
2. EMA, EMA/CPMP/BWP/268/95, Note for Guidance on Virus Validation Studies (Feb. 14, 1996).
3. EMA, EMA/CHMP/BWP/126802/2012, Guideline on the Adventitious Agent Safety of Urine-Derived Medicinal Products (May 26, 2015).
4. WHO, “WHO Guidelines on Viral Inactivation and Removal Procedures Intended to Assure the Viral Safety of Human Blood Plasma Products,” Technical Report, Series No. 924, 2004 Annex 4 (March 1, 2004).
5. ICH, Q5A(R1) Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human or Animal Origin, Step 4 version (1999).
6. P.W. Barone, et al., Nat Biotechnol. 38 (5) 563–572 (2020 ).
7. P.H. Ma, et al., J Virol 88 (12) 6576–6578 (2014).
8. EMA, EMA/CAT/80183/2014, Guideline on the Quality, Non-clinical and Clinical Aspects of Gene Therapy Medicinal Products (March 22, 2018).
Tareq Jaber, email@example.com, is associate director, and Anja Tessarz, Anja.Tessarz@crl.com, is associate director RGDB, SME Viral Clearance; both at Charles River Laboratories.
Vol. 35, No. 12
When referring to this article, please cite it as T. Jaber and A. Tessarz, “Viral Clearance Studies: Challenges and Beyond,” BioPharm International 35 (12) 18–21,33 (2022).