Viral Clearance Validation in Biologic Drug Manufacturing
Explore how our innovative fluorescent viral surrogates and tailored services integrate seamlessly into viral clearance workflows
Viral Clearance
Fluorecense
Viral Surrogates
Quantification
Ready Kits
References
Why is Viral Clearance Validation Necessary in Biologic Drug Manufacturing?
Figure 1: Overview of ICH Q5A(R2) pillars of viral risk mitigation.
When manufacturing mammalian-derived biologics, viruses pose a unique threat in that a single infectious particle has the ability to generate a contamination event along the process line, or worse, an illness in a patient. Where some viruses are produced endogenously by the host cell line, most others are contaminants to the production process and can be introduced through either animal-derived raw materials, contaminated cell banks, insufficient equipment sanitization, or direct human contact1–6. The chances of this happening are slim, but not impossible.
Given their ability to cause a range of potentially fatal illnesses (i.e., gastrointestinal, ophthalmologic, genitourinary, respiratory, and neurological7–11), adventitious viruses pose significant risk to patient safety. Therefore, when a contamination event occurs, manufacturers must completely shut down production and potentially recall products, risking medication shortage. It can take a facility weeks to months to come back online after a viral contamination event and can cost up to millions of dollars.
From a regulatory perspective, guideline ICH Q5A(R2) defines the “three pillars of viral risk mitigation” that biologic drug manufacturers must adhere to while developing their processes. Proof of compliance with these pillars is required in regulatory submissions for both clinical (Investigational New Drug, IND) and commercial use (Biologics Licensing Application, BLA)12–16. As seen in Figure 1, these three pillars comprise sourcing of non-animal derived products, regular in-process batch-to-batch testing of material to ensure the absence of viral particles, and viral clearance validation of the downstream purification process. Viral clearance validation is performed to confirm whether the purification process is able to clear significant concentrations of virus in the unlikelihood that a viral contamination goes undetected during upstream cell culture12,13. In essence, viral clearance validation acts as a “fail-safe”, by preventing adventitious viruses from reaching the drug substance stage of processing if present.
Current Viral Clearance Validation Practices, Standards, and Constraints
The current gold standard for viral clearance validation is a live viral spiking study. This workflow involves two main steps (see Figure 2). First, in-process load material (i.e., crude drug substance) is spiked with high concentrations of live virus and processed across the desired downstream unit operation. Second, virus particle concentration of the load and efflux pools are quantified. From this, the logarithmic reduction value (LRV) of virus across the unit operation is calculated as the difference in the logarithm of the infectious viral particle concentration between the load and efflux pools. LRV values are then employed in regulatory packages to demonstrate viral clearance capacity.
Figure 2: Workflow for live virus spiking study.
Model Virus Selection
Model virus panel composition depends on how one plans to use their clearance results as well as the host cell line implemented in their manufacturing process. For IND submissions, two-model virus studies are performed, employing one non-enveloped and one enveloped virus. BLA-bound studies are more complex, utilizing four model viruses (i.e., two non-enveloped and enveloped model viruses each). Regulatory agencies recommend that model virus panels are constructed according to the following characteristics: endogenously produced vs. adventitious, DNA vs. RNA genome, and enveloped vs. non-enveloped. In 201017, compiled viral clearance data from regulatory databases showed that IND submissions most commonly employed retrovirus (RNA, enveloped) and parvovirus (DNA, non-enveloped), and that BLA submissions typically utilized retrovirus, parvovirus, herpesvirus (DNA, enveloped), and reovirus (RNA, non-enveloped). These model viruses are still favored today as retrovirus can mimic endogenous retrovirus-like particles18, and parvovirus, herpesvirus, and reovirus can serve as general model contaminant viruses14. As for selection of the viral species themselves, that depends on which species are thought to most likely infect the host cell line during production. For example, when working with Chinese Hamster Ovary (CHO) cell lines specifically, Minute Virus of Mouse (MVM) is often chosen as the model parvovirus.
Viral Spiking Study Design
Regulatory agencies require at least two “viral clearance-dedicated” steps when developing downstream processes for mammalian-derived biologics: viral inactivation (VI) and viral filtration (VF). Therefore, spiking studies must at least be performed for these two steps to confirm sufficient viral clearance. Spiking studies across VI and VF are more complex. VI studies involve collecting multiple time-based samples to evaluate inactivation kinetics of enveloped viruses. VF experiments are designed to evaluate how differential pressure and “process pauses” impact viral clearance of smaller non-enveloped viruses. Though not required, viral clearance capacities of chromatographic steps are also evaluated as a way to claim additional viral clearance for the process. Though, this is only done for chromatography steps where at least 1 log PFU/mL clearance is expected14. In a regulatory format, chromatographic spiking studies are straightforward, involving collection of load and product pool aliquots for viral titer quantification, to calculate LRVs (and mass balances if desired). Complexity of chromatographic spiking studies generally depend on the drug’s stage in its product lifecycle. BLA-bound chromatographic clearance packages, for example, must include viral clearance data for both new and aged resin cases, as well as carry-over runs19. IND studies and general R+D work only need new resin data.
Viral Particle Quantification
Typically, a well-developed downstream purification process can claim anywhere between 10 and 25 log10 PFU/mL clearance17. At the bare minimum, the process must be able to clear at least 4.0 log10 PFU/mL for enveloped viruses, and at least 6.0 log10 PFU/mL for non-enveloped viruses12,13,16.
The current gold standard for infectious viral particle quantification involves plaque titer and TCID50 assays. Plaque titer assays determine lytic viral titer by quantifying plaques (1 plaque = 1 infectious viral particle per sample) formed in a monolayer culture of indicator cells incubated with virus-containing sample20,21. TCID50 assays determine the sample dilution required to produce observable morphological changes, assessed via optical microscopy, in 50% of the indicator cells22. Quantitative polymerase chain reaction (qPCR) is also regularly performed on VI and Protein A affinity chromatography samples to estimate the total amount of viral particles in each sample (i.e., the physical viral titer) for comparison to infective titer.
Current Constraints
For biologic drug manufacturers, working with live virus generates risk and complexity; mainly because industry labs are often only Biosafety Level (BSL)-1 certified, and located alongside their manufacturing facilities. To minimize contamination risk and have access to a BSL-2 lab space, live viral spiking studies are routinely performed off-site in collaboration with a third-party contract research organization (CRO). Between preparing the materials and onboarding the CRO, off-site spiking studies can take months to perform and cost hundreds of thousands of dollars ($100,000 (IND) – $400,000 (BLA))23. Further, the preservation and quantification of virus-containing samples14 also poses high risk of error and is not time efficient20,22. For the plaque titer and TCID50 assays alone, 14-28 days of error-free cell culturing are needed to allow for virus propagation, cytopathic effect, and plaque development14,20,22. Cumulatively, these downsides ultimately translate to higher drug prices for the patient and longer lead times for drug development and regulatory approval.
Viral Surrogate Technology
Current Viral Surrogate Landscape
Viral surrogates were recently developed to serve as a substitute for live viruses in early-phase research and characterization studies. They work by mimicking the external physicochemical characteristics of live virus particles (i.e., size, morphology, surface charge, hydrophobicity) while remaining noninfectious. Working with a noninfectious viral surrogate can add significant benefit to your workflow by allowing for the execution of spiking studies in-house, in a BSL-1 environment. This eliminates the need for intensive CRO collaboration, saving drug manufacturers months of time and hundreds of thousands of dollars, helping to get regulatory submissions out the door faster, at a fraction of the cost.
Two general types of viral surrogates are currently employed in viral clearance: viral-like particles (VLPs) and bacteriophages. However, because of key differences in their biophysical characteristics compared to live viruses, they have not yet been applied in regulatory formats. Futher, viral surrogate quantification assays are currently lower resolution than those used in live virus studies, and remain error-prone, highly involved, and time-consuming. This is mainly because formulating a quantification strategy for viral surrogates is deceptively more difficult, as they cannot natively contain DNA or RNA (otherwise they would be infectious). Excluding bacteriophages, viral surrogates cannot rely on highly sensitive, more traditional forms of quantification like PCR or TCID50 as a traditional virus system could. This results in more complex quantification and reliance on alternative, less familiar methods. In many contexts, current viral surrogate strategies offer significant improvements over live viral study workflows. However, we believe there is still room for improvement in the space.
How we stack up:
Figure 3: An overview of the current viral surrogate landscape.
How the Ready Kits Work
Application in Viral Clearance
Quantification via Dead-End Flow FVS Capture
Our website may use cookies to enhance your browsing experience and collect information about how you interact with our site. You can adjust your browser settings to refuse cookies or alert you when cookies are being sent, but some features of the site may not function properly without cookies.
References
We reserve the right to update or change this Privacy Policy at any time. Any changes will be posted on this page, and the effective date will be updated accordingly. We encourage you to review this Privacy Policy periodically for any updates.