Process understanding and careful assessment of risks are essential in developing viral clearance programs.
By Rita C. Peters
Drug products based on live cells have an inherent risk for viral contamination that could cause serious harm or death to patients. When applying for regulatory approval, a drug license holder must demonstrate that its proposed manufacturing process can remove or inactivate potential viral contaminants.
Viral clearance studies assess the capability of a downstream process to remove or inactivate potential contaminants and are based on a scale-down model of a biopharmaceutical production process. The studies must be designed to evaluate potential viruses, be representative of the production process, meet regulatory requirements, and provide reportable data. In addition, the testing should be conducted under “worst-case” conditions.
Viral clearance study elements
“The scale-down process used in the spiking study must be representative of the full-scale process and care must be taken to demonstrate the validity of the scale-down model using appropriate model viruses,” says Kate Smith, principal scientist, global operational development services, MilliporeSigma. “The study should be carefully designed to ensure the capacity to remove or inactivate virus is not over-estimated, applying ‘worst-case’ parameters to individual steps, where known.”
The key elements of a viral clearance validation are choice of process steps, viruses, points of sample withdrawal, and the correctness of scaling, says Gustav Gilljam, client project manager, Vironova Biosafety. “The process steps chosen must represent different mechanisms in virus inactivation/removal to be included. The viruses tested should include both enveloped and non-enveloped viruses, and both RNA and DNA viruses with different physical and chemical properties.” He also notes that the studies should be conducted under worst-case conditions and performed according to good laboratory practice.
Developing a test protocol, including the selection of viruses, is crucial. The model viruses used, and the number of steps investigated for viral clearance capacity, should be related to the risk assessment and product-specific regulatory guidance, says Horst Ruppach, director, viral clearance and virology at Charles River. “Pre-tests like cytotoxicity and interference assays are essential to prove the validity of the assays used for viral quantification,” he explains. “The product- and step-specific adjustment of virus spiking, sample treatment, and assay sensitivity ensure the viral clearance potential can adequately be demonstrated.”
Drug license holders often turn to contract testing laboratories for this specialized testing; planning and communication are key elements. “Studies should be planned enough in advance to allow time to determine the scope and essential parameters of the study, to obtain the necessary materials for the study, and to work with your testing partner to schedule the study,” says Katherine F. Bergmann, manager, viral safety and clearance services, Eurofins Lancaster Laboratories. “The key parameters that determine the scope of the study are the clinical stage and indication of the product and the nature and origin of the source material.”
What to test, and when
Viral clearance studies typically are conducted at two phases of product development. “The early-stage study demonstrates the general viral clearance capacity, while the late-stage study typically demonstrates the robustness of the clearance capacity,” says Ruppach. “The extent of both depends on the product type and the development phase.”
While only one or two viruses and a minimum number of samples are tested in early clinical phase, says Bergmann, four or five viruses are typically tested for products approaching or in commercial manufacturing. “Additional samples are evaluated in order to determine mass balance,” she says. “In addition, expanded ranges of critical operating parameters may be evaluated, and column cleaning and carry-over must be evaluated.”
In testing prior to Phase III, Gilljam notes, “Reduction of virus infectivity is the most important measure, but various methods to detect viral genome copies or physical particles could be a complement. The manufacturing limits of parameters in these steps that might have an impact on the virus reduction should be challenged in the virus validation studies prior to Phase III, to show the robustness of the steps.”
Smith notes the key areas of viral safety testing. Raw materials and cell substrates must be characterized using molecular, in-vitro, and in-vivo testing strategies, she says, and cell banks and end-of-production cells are tested to identify species of origin, confirm expression construct stability, and to demonstrate the absence of potential bacterial and viral contamination. “Additional routine bulk harvest testing and the viral clearance study complete the tripod of viral safety testing,” she says.
Challenges in study design
The multi-step nature of bioprocesses—and the viruses themselves—present challenges to the development of viral clearance studies and limit their effectiveness.
“All steps in a viral clearance study must be orthogonal; they must clear virus by independent mechanisms,” says Bergmann. “For example, if one step inactivates virus due to low pH, then a second step that uses a low pH buffer may not also be included, since virus that escapes inactivation by one low pH step is likely to be resistant to a second low pH incubation. Achieving a satisfactory clearance level can be limited by the virus titer, by cytotoxicity, or by viral interference,” she explains. “These limitations can be addressed by careful study design to optimize the clearance available from each step.”
The European Medicines Agency lists potential limitations in a guideline (1), notes Smith. “Test sample composition can limit assay sensitivity and reduction factors obtained where large dilutions are required to alleviate cytotoxic effect on indicator cell lines,” she says. “Virus spike quality can influence the operation of key steps; for example, higher purity virus spikes are required for virus reduction filters to minimize virus induced flux decay and achieve the target filter capacity.” International Council for Harmonization (ICH) Q5A (2) also lists limitations of viral clearance studies.
Ruppach and Gilljam note that the use of virus models presents some limitations. “Relevant viruses may be difficult or impossible to produce in high concentrations. Therefore, model viruses, preferably of the same genus or family, are used as substitutes,” says Gilljam.
“Even though few model viruses represent the biophysical characteristics of a broad range of mammalian viruses of different virus families, they are still models prepared and kept under laboratory conditions. They do not perfectly mimic the characteristics of related wild-type viruses,” says Ruppach. “This is of specific concern for biopharmaceuticals directly isolated from human or animal tissues, like human plasma products. On the other hand, the laboratory model viruses have a great chance of contaminating bioreactors using continuous cell lines.”
Gilljam identified other challenges. “Due to the volume tested in the viral clearance study and that a dilution of the sample is needed to avoid any cytotoxicity to the indicator cells and the interference with the ability of the indicator cells to be infected with the virus, it is not possible to analyze the entire volume for the presence of infectious virus,” he says. “There is always a lower limit of detection, so the virus infectivity may be greatly reduced, but never to zero. Not all viruses are known. We will find what we look for but may miss the unexpected.”
The relevance of the model virus depends on the virus step analyzed, Ruppach notes. “Virus retentive filtration is based on size exclusion, and a model of a specific size will represent all viruses of the same or greater size. But removal with chromatography steps is based on the virus envelope characteristics, which can differ among viruses of the same virus family and even among different strains,” he explains.
Another limiting factor is the relevance of the downscale model. “In some cases, it’s easy to downscale the manufacturing scale but in other cases a 1:1 downscale isn’t possible,” Ruppach says.
As in other areas of biopharmaceutical development, a risk assessment can define potential risks of viral contamination—and the need for viral clearance—in different phases of a production process.
Risk assessment defines the viruses of risk, the type of and how many model viruses should be analyzed in the viral clearance study, and the overall reduction needed, says Ruppach. “In general, a high and robust viral clearance capacity is a much more efficient measurement to reduce viral risk than any extensive viral testing program; in fact, it can significantly reduce the testing on viruses.”
Assessments should consider the entire production process and any changes.
The source of starting materials and raw materials used in production are of primary concern, says Bergmann, and a risk assessment identifies which viruses are likely to be contaminants and the levels of contamination that could potentially exist. “This analysis is relatively simple for biologicals produced in well-characterized cells in culture but can be much more complex for products derived from uncharacterized cells or from animal materials (e.g., tissue),” she said. “Understanding the risks of viral contamination leads to the choice of viruses selected for a study, as well as the level of virus clearance required, to assure a safe product.”
Assessments should consider the entire production process and any changes. “The impact on the validity of the viral clearance data by changes to the process, such as facility, procedural, process, and raw materials must be assessed,” says Smith. “A detailed risk assessment covering all aspects of the manufacturing process demonstrates an understanding and control of the process and provides a framework to evaluate the impact of each change both on the affected unit operation and those following. An informed decision can then be made to determine the scope of revalidation required.”
Keeping pace with biopharma advances
While the primary guidance documents—EMA/CHMP/BWP/268/95 and ICH 5QA—are 20 years old, they are still adequate for determining the background and principles for viral clearance, says Ruppach. There is space for adaptions based on the experiences made since the guidances were issued, he adds.
Bergmann concurs and notes that while these guidance documents work well for monoclonal antibodies and therapeutic proteins, “use of the guidelines for novel types of products can be challenging” and revisions of regulatory guidance documents need to address these product types.
Advances in production technology also can present complications for the viral clearance studies. “A lack of appropriate scale-down models and strategies to support changes in industry practices such as continuous processing can restrict the design of the viral clearance study,” Smith says. “As new markets open up to the industry, greater visibility of requirements supporting biosimilars, validation of continuous processes, and global submissions would be beneficial.”
1. EMA, Notes for Guidance on Virus Validation Studies: The Design, Contribution and Interpretation of Studies Validating the Inactivation and Removal of Viruses, CHMP/BWP/268/95, London, Feb. 14, 1996.
2. ICH, Q5A (R1) Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human or Animal Origin, Step 4 version (ICH, 1999).