Ensuring viral safety in advanced therapies

As the field of advanced therapies continues to expand, the complexity of ensuring viral safety in gene and cell therapy manufacturing has become a central concern. Viral clearance (VC) studies, long a cornerstone of biologics production, are now being reevaluated and adapted for genetically engineered viral vectors and vector-derived products. The latest revision of the ICH Q5A guideline offers new clarity while also underscoring the need for case-by-case risk assessments and tailored approaches.

A regulatory imperative

The updated ICH Q5A (R2) guidance states that VC studies “should be performed to determine virus reduction factors for the relevant step(s) of the production process” when appropriate. While this language allows for flexibility, it also signals that VC studies are expected for late-phase development. The extent and nature of these studies, however, depend on the specific risk profile of each product.

This shift in regulatory expectations means manufacturers must revisit the foundational principles of viral contamination control, which were originally developed for monoclonal antibodies and recombinant proteins, and apply them to the unique challenges of gene therapy products.

Sources of viral contamination

Contamination risks in gene therapy manufacturing can arise from multiple sources:

• Cell lines: Commonly used substrates such as BHK, HEK293, and Sf9 cells carry potential for endogenous or latent viruses. For example, BHK cells may harbor retroviruses, HEK293 cells may contain adenoviruses, and Sf9 cells are associated with rhabdoviruses.
• Raw materials and reagents: Contaminants like bovine viral diarrhea virus (BVDV) from serum or minute virus of mice (MVM) from media can enter during cell culture, formulation, or purification.
• Manufacturing environment (including operators): Cross-contamination risks are heightened in multi-product facilities, where gene therapy products may be produced alongside other biologics.
• Helper viruses: These are used in the production of viral vectors and must be considered in VC studies. Some examples for helper viruses they are commonly used are baculovirus, herpes simplex virus type 1 (HSV-1), adenovirus type 5 (AdV5).

Selecting viruses for clearance studies

ICH Q5A (R2) emphasizes the inclusion of model viruses that represent adventitious, endogenous, and production viruses. For the latter a new category, Case F, was introduced to address helper viruses and viral vectors used in recombinant protein or virus-like particle (VLP) production.

Examples relevant for gene therapy manufacturing include:

• Baculovirus/Sf9 system: Sf9 cells fall under Case C due to the presence of rhabdovirus. While direct testing with rhabdovirus may be impractical, vesicular stomatitis virus (VSV) is commonly used as a surrogate. Baculovirus, as a helper virus, is addressed under Case F.
• BHK-21/helpervirus expression system: BHK-21 cells are covered under Cases B and D, while their associated helper viruses, adenovirus and herpesvirus, are addressed in Case F.
• Transient transfection systems using HEK293 cells: HEK293 cells even though they are Case A cells, meaning no virus-like particle nor retrovirus-like particle were found, and no production virus is being used for the transient transfection, a risk assessment is essential, and a VC study is expected. The latter should use nonspecific model viruses that span a range of characteristics: enveloped vs. non-enveloped, RNA vs. DNA, and varying resistance levels.

Log reduction expectations

Unlike monoclonal antibodies, gene therapy products often lack robust VC steps. ICH Q5A (R2) recommends implementing two distinct VC steps, ideally one targeting non-enveloped viruses. However, it acknowledges that viral vector processes may not achieve the same efficiency as recombinant protein production.

Effective VC steps typically demonstrate reproducible reductions of 1 to 3 log₁₀, with higher reductions contributing more significantly to overall safety. Importantly, there is no mandated 4 log₁₀ reduction target for viral vector products.

Viral clearance challenges

Gene therapy products face unique constraints in downstream processing: 

• Limited purification steps: Unlike monoclonal antibodies, gene therapy products often lack multiple polishing steps.
• Product nature: Since the product is itself a virus, traditional VC methods like 20nm filtration and detergent treatments are often unsuitable as the product itself would be retained by the filter or in case of lentivirus and herpesvirus systems for example is sensitive to detergents. The virus removal potential of chromatography steps based on charge separation can also be limited, as some viruses may share similar properties with the viral vector product and thus co-elute (e.g. parvovirus co-elution with the AAV product).
• Limited test material: having very limited material for testing available (often one batch required to perform VC study) is very costly. Providing enough material for VC study performance is one of the major challenges for this kind of product. Co-spiking approach might be a great solution for this challenge.

Common VC study steps

Despite these limitations, certain downstream steps have shown promise in VC studies, particularly for AAV products. Data from Charles River Labs highlights the following:

• Affinity chromatography: For mAbs and recombinant proteins, affinity chromatography for product purification typically results in moderate virus reduction, with log reduction values (LRVs) ranging from below 1 log₁₀ to 4 logs on average. In studies involving AAV products, major viruses such as MuLV, adenovirus, Reo, and VSV showed variable clearance, with many runs achieving 3–4 log₁₀ reductions.
• Anion exchange chromatography (AEX): AEX is considered one of the most robust VC steps. In CHO-derived products, over 65% of runs achieved reductions above 4 log₁₀. AAV products showed similar results, with tight LRV ranges and consistent performance across multiple viruses.
• Viral filtration: While traditional 20nm filters are unsuitable for gene therapy, for AAV systems larger filters (>30nm) can be evaluated using model viruses like SV-40 (45nm), FCV (35–40nm), and BVDV (50nm). These studies support the principle of size-based exclusion, though smaller viruses like parvoviruses (18–20nm) remain a challenge. Overall, viral filtration has shown average reductions above 4 log₁₀ with minimal residual infectivity for viruses ≥ 35 nm in size.
• Detergent treatment: Detergents like Triton X have historically been used to inactivate enveloped viruses. However, due to environmental concerns, alternatives are being explored. New solvents and detergents have shown promising results, though effectiveness depends on concentration, incubation time, and temperature. Each product requires tailored conditions to optimize viral inactivation.

Alternatives to VC (sourcing and testing)

Given the constraints of VC in gene therapy, manufacturers must adopt a multi-pronged approach to ensure viral safety:

• Raw material control: Use of well-characterized cell banks and virus seeds reduces contamination risk.
• Animal-derived materials: Manufacturing processes should avoid the use of human- and animal-derived raw materials such as human serum, bovine serum, and porcine trypsin.
• Media treatment: Techniques such as gamma irradiation, virus filtration, high-temperature short-time processing, and UV-C irradiation can mitigate viral risks.
• Closed processing systems: Minimizing human and environmental exposure helps prevent contamination.
• Testing: Testing for viral contamination must be integrated at critical points of the manufacturing process, starting from raw materials through to the final drug substance.

Looking ahead

As regulatory frameworks evolve and new therapies emerge, VC strategies must adapt. The goal remains clear: to identify and validate virus reduction factors that ensure the safety of gene therapy products. By combining rigorous risk assessments with innovative clearance methods, manufacturers can meet regulatory expectations and deliver safe, effective treatments to patients.