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The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) provides guidance on the testing and evaluation of viral safety of biotechnological products derived from characterized cell lines of human or animal origin through its harmonized guideline ICH Q5A (1). The latest revision, released for consultation in October 2022, maintains the key principles of previous versions while introducing key changes in response to important advances in the field. Those advances are covered in new sections that reflect improvements to current scientific knowledge. They include new product types that are amenable to viral clearance, including viral-vector–derived products; considerations for continuous manufacturing; and the use of prior knowledge (PrK) for modular validation and resin-reuse studies. Also discussed are new molecular analytical methods, particularly next-generation sequencing (NGS). The aims of this update are to reduce the need for product-specific validation efforts and to provide flexibility for the testing of already well-characterized rodent cell substrates.

ICH Q5A’s second revision (R2) completed the public consultation phase in February 2023. Although minor changes could be made to the final document in response to comments, the essence of this revision is likely to remain as currently proposed. Thus, it is well placed to support the implementation of advances in viral safety based on recent published scientific advances. Below, we review and summarize the updated topics and discuss their impact on viral-safety testing and evaluation.

Viral Vectors and Related Products
Since the release of ICH Q5A’s first revision (R1) in the late 1990s, new and advanced cell-based production platforms have been developed for manufacturing new product types. Products manufactured using such platforms include genetically engineered viral vectors and viral-vector–derived products, both dependent on and independent of helper viruses. For such products, viral clearance without negative product effects has become increasingly important (2). For some time, it has been the practice of regulatory authorities worldwide to require evidence of that at least at the time of commercial licensing. Products such as virus-like particles (VLPs) and protein subunits made using baculovirus, herpes-simplex virus, or adenovirus production systems — as well as nanoparticle-based vaccines and viral vectors such as adenoassociated virus (AAV) — now are considered in the new Annex 7 to ICH Q5A.

Manufacturing platforms used for those products have the potential to introduce viral-contamination risks over and above the general viral-safety considerations for biopharmaceutical products. The expression systems used include a host of less-common cell substrates, including insect cell lines, for which general substrate characterization knowledge is not as extensive as it is for the standard eukaryotic cell workhorses — e.g., Chinese hamster ovary (CHO) cells — used in most biomanufacturing processes. For viral-vector and other products manufactured using viral-vector platforms, the potential for contamination with replication-competent viruses also needs to be considered. The revised ICH Q5A advocates a risk-based approach to ensuring viral safety of these products that should address the associated raw materials, options for viral testing at appropriate manufacturing steps, and removal/inactivation of both adventitious and helper viruses during downstream processing.

In addition to the standard approach of testing for adventitious viruses while characterizing master cell banks (MCBs), working cell banks (WCBs), and cells at the limit of in vitro age (LIVCA), product developers need to consider aspects such as the presence of residual helper viruses and replication-competent viruses. Similarly, the guideline recommends testing for adventitious/endogenous viruses as well as helper and replication-competent viruses in virus seeds, unprocessed bulk (harvest) material, and drug substance (3). For products produced by insect cells, it also suggests testing for certain known intracellular contaminants such as insect rhabdovirus. One challenge that developers can face in this regard is testing viral seeds that could propagate through indicator cells. Details of the testing approach depend on the specific viral vector and viral-based production system used as well as on test results for the cellular substrate that can indicate residual retroviral activity, for example.

If viral-clearance studies can be conducted, then they need to demonstrate the capacity of a manufacturing process to remove and/or inactivate potential viral contaminations at relevant manufacturing steps. For viral vectors and related products, the revised guideline highlights specific considerations for determining whether and how such studies should be conducted. Initially, the physicochemical characteristics of viral vectors and viral-vector–derived products determine whether an approach is feasible and how virus clearance can be applied within the purification operation. When studies are conducted, virus-clearance validation normally must include model viruses that represent the most likely adventitious, endogenous, and (if possible/necessary), relevant helper viruses that can be expected for a given product. To be representative, model viruses need to be given careful consideration and the choices scientifically justified to regulators.

Developers also need to consider the suitability of viral-inactivation and filtration steps for the types of viral vectors used. Standard technologies include detergents and/or solvents for nonenveloped viral vectors and large, virus-specific, retentive filters for small vectors such as AAVs. It is important to remember that helper viruses are process-related impurities, so their adequate clearance must be demonstrated. Experience shows that although high virus log-reduction factors can be achieved regularly for such products, those factors are not comparable to the clearance observed with recombinant proteins. Therefore, viral clearance only contributes to product safety. It must be underpinned by a full-spectrum control strategy for adventitious agents including, for example, closed processing, material controls, and in-process and product testing.

Continuous Manufacturing
For many traditional biotechnology products, the shift from conventional batch processes toward continuous manufacturing (CM) is gaining momentum. CM is used to produce and/or process products without interruption between batches so that raw materials, process intermediates, and starting materials are fed continuously into a manufacturing process while products are discharged continuously — or in a series of pulses — throughout an operation period (4). In this type of biomanufacturing, multiple process steps are controlled automatically, thereby simplifying an overall operation and reducing process-associated risks. The advantages of CM include increased efficiency and productivity with associated economic benefits.

The revised guideline recognizes that technical aspects of viral-safety control for CM necessarily will differ to some extent from those for batch processing. Monitoring, detection, and removal of viruses are affected by system dynamics, the monitoring frequency, and both system start-ups and shutdowns. Advanced process controls, process models and their validation, and continuous process verification also can influence viral-control strategy in CM operations. Therefore, the viral-safety controls for a CM process need to be designed with all those aspects in mind.

Fluctuations of different parameters in a CM system, including protein-product concentration, can affect both the validity of in-process testing and the design of viral-clearance studies. ICH Q5A(R2) discusses key considerations in this respect. They include the extent to which a process is run continuously and whether only some production steps are directly connected. Such concerns feed into assessments of the risk that upstream failures could put downstream operations in jeopardy — a risk that also is influenced by the types of connections between unit operations.

The guideline acknowledges that it is feasible to use PrK based on batch processes for evaluating unit operations, especially when a process is a series of “minibatch” pulses rather than one continuous stream. In some situations, continuous processes can be treated as a series of batch-mode processes, validation for which is based on small-scale batch models covering potential extremes in parameters such as transient protein concentration and column backpressure. Appropriate process monitoring and sampling strategies are needed to detect inadvertent disturbances and adventitious virus contamination, taking into account the potential effects of fluctuations in raw-material attributes, flow rate, operational loading capacity, and multicolumn cycling. The guideline also discusses other viral-safety aspects of CM, such as appropriate sampling points and risks associated with long-term cell culture. The latter can include potential fluctuations in the levels of endogenous retroviruses and other viral contaminations.

Virus-clearance studies for processes such as chromatography, low-pH treatment, solvent/detergent inactivation, and virus filtration also require an adapted approach. Note that ICH Q5A(R2) acknowledges a number of settings and circumstances under which viral-clearance testing for a continuous process can use scaled-down batch models. For example, clearance studies using batch-based small-scale models can be an option for chromatography unit operations and are discussed in the guideline. For repeat-use systems (e.g., multicolumn processing of successive subbatches), sponsors may use a well-justified scale-down model based on a batch process. Simultaneous validation of two or more connected unit operations also is possible when an entire unit operation is intended for viral clearance. Study design is affected by equipment considerations and unit integration.

For low-pH and solvent/detergent inactivation processes, CM validation using batch operations also could be justified if control of relevant dynamic process parameters is ensured: pH, concentration of solvent/detergent, homogeneity and mixing conditions, temperature, and time. For virus-filtration steps, CM validation using batch processes can be appropriate when settings or ranges are established for relevant parameters (e.g., transmembrane pressure) that can affect viral clearance — and where those ranges are well justified. Additionally, process controls must be defined such that filter changes and postuse integrity tests can be performed without interruption of CM, and process designs should include optional diversion of material to a backup filter in case of primary filter failure or blockage.

New Molecular Test Methods
Global regulatory bodies now encourage biopharmaceutical developers to use new test methods based on NGS and polymerase chain reaction (PCR), for example, to align with the initiative of reducing animal use in drug testing — the 3R principle of replacement, reduction, and refinement (5). The revised ICH Q5A provides more details on considerations for alternative analytical methods.

Nucleic-acid–amplification techniques (NATs) such as PCR-based analytical methods are used singly or in a multiplex format to detect specific virus sequences or measure model virus levels in clearance studies. Such molecular methods are effective tools for detection of specific viruses and can be used to supplement cell-based assays that are limited by interference. NATs also can be adapted for detection of a broad range of viruses, although specificity of results in such cases might be reduced. Whichever approach is taken, NAT-based methods need to be qualified or validated appropriately for their intended use.

Advanced molecular methods such as NGS can detect a broad range of different viruses. Because of the sensitivity and breadth of virus detection capability with such methodologies, the guideline recommends their use to reduce both testing time and the use of laboratory animals. Developers are encouraged to provide a complete validation package to support their use of NGS, including method validation and assay or matrix-specific qualification. In addition, suitable standards and reference materials should be used for assay qualification and validation, including available reference-virus reagents with distinct physical, chemical, and genomic characteristics. When applying NGS, critical steps include sampling and sample processing, efficient nucleic acid extraction and library preparation, sequencing platform selection, and comprehensive bioinformatics analysis.

Overall, the guideline encourages developers to use NGS as a replacement for

• in vivo tests with broad virus-detection capabilities for known and unknown or unexpected virus species
• hamster-antibody production (HAP), mouse-antibody production (MAP), and rat-antibody production (RAP) tests.

NGS is considered to be useful particularly for characterization and testing of cell substrates and cell banks, viral seeds, and harvests. Because NGS has a complex workflow, however, the guideline encourages manufacturers to discuss method validation and data submission with regulatory authorities. ICH Q5A(R2) signals a clear commitment of international regulators to accept NGS as a key method for virus detection that should be used as the biopharmaceutical industry progresses.

Prior Knowledge: Including “In-House” Experience
The term prior knowledge has been used in several ICH guidelines without a formal statement of its meaning (6–10). The draft revision of Q5A(R2) contains a welcome definition. Furthermore, this draft guidance provides several examples of how PrK can be applied to evaluation of steps involved in viral clearance and reduction, which thereby potentially allows for alternative viral-clearance validation strategies. For example, a footnote to Table A-1 of the guideline suggests that PrK based on scientific principles can be applied to validating the performance of small virus-retentive filters — e.g., by using parvovirus instead of larger viruses as a worst-case model, an approach that makes sense scientifically. Parvoviruses facilitate handling and filter throughput, making such an approach more practical logistically.

A new Annex 6 to the revised guideline provides useful details for developers applying PrK from their own product development and experience to similar products or platforms (internal/in-house PrK) for reducing the amount of product-specific data needed. If PrK is leveraged in lieu of product-specific experiments, it should be accompanied by a holistic viral risk assessment for a given medicinal product. Factors to consider that can influence the potential quantity of infectious particles in the product include information gained from the cell-substrate and raw-material characterization and the overall viral-clearance strategy. Adding PrK-related content to the guideline has been a welcome step toward flexibility regarding the provision of data from such supportive studies that could provide time savings for the industry.

Prior Knowledge for Clearance Evaluation and Validation
Viral clearance generally is evaluated in experiments based on spiking different viruses into product-specific in-process material from each downstream processing step. When a biomanufacturer is using a process that has been established and well characterized for a similar product (using the same platform technology), then clearance data generated for previous products could be applicable to a new product. This is called in-house knowledge in the European Union; in the United States, it is called modular clearance. The applicability (“representativeness”) of such PrK for a specific process step should be justified clearly. Whether based on external or in-house experience, PrK should include

• a good understanding of the mechanism underlying virus clearance
• all process parameters affecting viral clearance
• the influence of interactions between viruses and biological products
• potential interference based on composition of some process intermediate(s) with viral clearance.

External PrK based on published data can be useful in understanding and predicting the clearance potential of a given manufacturing step. For example, it can help developers define critical process parameters (CPPs) and predict worst-case limits for testing. The mechanism underlying virus clearance is well understood for many unit operations based on years of scientific evaluation described in published literature, and in many cases it does not differ among products. However, ICH Q5A(R2) suggests limiting the role of externally published reduction factors for specific products to support in-house knowledge and process-specific validation efforts. Application of external published reduction factors requires

• accompanying evidence that demonstrates sufficient comparability of the external and in-house processes for manufacturing their respective products
• comparability of process intermediates
• assurance that product-specific attributes do not affect virus reduction.

Changes to a biomanufacturing process during product life-cycle management can affect virus-clearance efficacy and could be evaluated using internal PrK and a platform concept. If PrK from other products cannot be extrapolated to a specific product, and/or if the platform concept no longer can be applied, then product-specific viral-clearance studies must be performed.

Annex 6 to the ICH Q5A(R2) guideline provides case studies of specific unit operations in which PrK (including in-house experience from other products) could be used to claim a reduction factor for a new product made using the same manufacturing platform. In general, the referenced unit operations are robust and well understood in the industry. A virus-clearance claim for a new product based on PrK (including in-house experience) should include discussion of all data available and the rationale to support the platform validation approach. Sponsors could provide part of the PrK used to reduce product-specific validation requirements in the form of a comparison of the new product and its manufacturing process with other in-house products, related process conditions, and product intermediates. Thus, process steps dedicated to virus clearance (e.g., inactivation by solvents/detergents, low-pH treatment, and removal by viral filtration) are suitable to a platform validation approach. The revised guidance provides examples for applications of PrK — such as for xenotropic murine leukemia virus (X-MuLV) inactivation/removal by detergent or low pH or for parvovirus removal by virus filtration — and describes process parameters of the different inactivation/removal steps and their criticality in relation to potential step performance.

Chromatography Media
Earlier versions of ICH Q5A focused on the concern that over time, and after repeated use, viral-clearance capability can diminish for chromatography columns and other components used in purification processes. Chromatography media/resin lifetime use should be established, and CPPs that affect viral clearance should be defined. Since the initial release of ICH Q5A in 1998, multiple published scientific studies and industry databases have contributed to PrK (11–13). Thus, R2 provides some flexibility on these requirements for well-known chromatography media such as protein A affinity and anion-exchange resins.

For protein A affinity chromatography, PrK indicates that virus removal is not affected — or even can increase slightly — in used (e.g., end-of-life) media. Therefore, product-specific studies are not expected for used resins. That has been acknowledged by regulatory authorities (14), but now it is stated explicitly in ICH Q5A(R2). Resin life has been studied less for other chromatography types, such as anion exchange. Accordingly, repeated resin use for those chromatography types requires equivalent in-house experience and a detailed justification to replace product-specific viral-clearance studies of end-of-lifetime media.

Flexibility on Testing of Well-Characterized Cell Substrates
PrK also can be applied to provide some flexibility in requirements for well-characterized cell lines. For instance, in a case study for calculating per-dose virus-safety factors, Annex 5 states that a safety margin of <10^–4 particles/dose for retroviral-like particles from CHO cells is acceptable for recombinant proteins if in vitro testing fails to identify the presence of infectious retroviruses. That supplants a previous case study from the 1999 version of this guideline, in which the target safety margin was implied to be <10^–6 particles/dose. Although that was intended only as a case study, many industry and regulatory experts had interpreted the <10^–6 particles/dose to be a regulatory rule.

In addition, the requirements for in vivo adventitious-agent testing for well-characterized lines such as CHO cells, NS0 murine myeloma cells, and SP2/0 murine lymphocytes have been reduced significantly based on cell-line history, PrK, and on other risk-based considerations. PrK now can include prior in vivo virus testing or NGS testing of the untransfected parental cell line alongside demonstrated control of the derivation of the MCB from those parental cells. PrK regarding virus-safety testing of other MCBs derived from the same parental cell bank (according to the same protocol) also should be considered.

In vivo testing also is generally unnecessary for the first WCB or subsequent WCBs from that MCB if they are prepared under approved, controlled conditions. Similarly, for cells at the LIVCA, such testing may be unnecessary according to PrK and other risk-based considerations. If a residual risk is determined, then retention of the test or replacement with a molecular method for broad virus detection (e.g., NGS or PCR) could be applied to detect viruses that might be introduced during establishment of the MCB or during cell culturing at the LIVCA stage.

A Solid Update
The released draft of ICH Q5A(R2) provides comprehensive and up-to-date guidance on viral-safety evaluations for biotechnological products derived from cell lines of human and animal origin. It has been kept sufficiently flexible to accommodate novel approaches and includes several examples and statements that clarify considerations in the light of new products, production types, emerging science, and PrK.

The guideline continues to emphasize the value of a multipronged approach to viral safety, including comprehensive characterization and screening of cell substrates as well as raw and starting materials to identify potential viral contaminants. It also encourages establishment of appropriate testing programs for adventitious, endogenous, and helper viruses in unprocessed bulk drug substance. This guideline stresses the need for careful design of viral-clearance studies, details of which depend on the type of biotechnological product that is manufactured, using different methods of virus inactivation and removal to achieve maximum viral clearance. The details of how to design those studies and achieve these goals have been updated based on extensive experience among companies and regulators over the 25 years since the first Q5A revision.

Altogether, we find ICH Q5A(R2) to be a successful revision that incorporates many important new developments brought about by scientific progress in the field of viral safety since the release of the Q5A(R1). We believe that, when it is finalized, the changes will have made this important guideline fully able to address the viral-safety concerns of the 21st-century biopharmaceutical industry.

References
1 ICH Q5A (R2). Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human or Animal Origin. International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use: Geneva, Switzerland, 29 September 2022; https://database.ich.org/sites/default/files/ICH_Q5A%28R2%29_Step2_draft_Guideline_2022_0826.pdf.

2 Barone PW, et al. Viral Contamination in Biologic Manufacture and Implications for Emerging Therapies. Nature Biotechnol. 38, 2020: 563–572; https://doi.org/10.1038/s41587-020-0507-2.

3 Ma H, et al. Identification of a Novel Rhabdovirus in Spodoptera frugiperda Cell Lines. J. Virol. 88(12) 2014: 6576–6585; https://doi.org/10.1128/JVI.00780-14.

4 ICH Q13. Continuous Manufacturing of Drug Substances and Drug Products. International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use: Geneva, Switzerland, 16 November 2022; https://database.ich.org/sites/default/files/ICH_Q13_Step2_DraftGuideline_%202021_0727.pdf.

5 EMA/CHMP/CVMP/JEG-3Rs/450091/2012. Guideline on the Principles of Regulatory Acceptance of 3Rs (Replacement, Reduction, Refinement) Testing Approaches. European Medicines Agency: Amsterdam, The Netherlands, 2012; https://www.ema.europa.eu/en/documents/scientific-guideline/guideline-principles-regulatory-acceptance-3rs-replacement-reduction-refinement-testing-approaches_en.pdf).

6 ICH Q2(R2). Validation of Analytical Procedures. International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use: Geneva, Switzerland, 1 November 2005; https://database.ich.org/sites/default/files/ICH_Q2-R2_Document_Step2_Guideline_2022_0324.pdf.

7 ICH Q8(R2). Pharmaceutical Development. International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use: Geneva, Switzerland, 1 August 2009; https://database.ich.org/sites/default/files/Q8%28R2%29%20Guideline.pdf.

8 ICH Q10. Pharmaceutical Quality System. International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use: Geneva, Switzerland, 4 June 2008; https://database.ich.org/sites/default/files/Q10%20Guideline.pdf.

9 ICH Q12. Technical and Regulatory Considerations for Pharmaceutical Product Lifecycle Management. International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use: Geneva, Switzerland, 20 November 2019; https://database.ich.org/sites/default/files/Q12_Guideline_Step4_2019_1119.pdf.

10 ICH Q14. Analytical Procedure Development. International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use: Geneva, Switzerland, 24 March 2022: https://database.ich.org/sites/default/files/ICH_Q14_Document_Step2_Guideline_2022_0324.pdf).

11 Mattila J, et al. Retrospective Evaluation of Cycled Resin in Viral Clearance Studies — A Multiple Company Collaboration. PDA J. Pharm. Sci. Technol. 73(5) 2019: 470–486; https://pubmed.ncbi.nlm.nih.gov/31101706.

12 Ajayi OO, et al. An Updated Analysis of Viral Clearance Unit Operations for Biotechnology Manufacturing. Curr. Res. Biotechnol. 4, 2022: 190–202; https://doi.org/10.1016/j.crbiot.2022.03.002.

13 Brorson K, et al. Identification of Protein A Media Performance Attributes That Can Be Monitored As Surrogates for Retrovirus Clearance During Extended Re-Use. J. Chromatogr. A 989(1) 2003: 155–163; https://pubmed.ncbi.nlm.nih.gov/12641291.

14 EMA/CHMP/BWP/187162/2018. Meeting Report: Joint BWP/QWP Workshop with Stakeholders in Relation to Prior Knowledge and Its Use in Regulatory Applications. European Medicines Agency: Amsterdam, The Netherlands, 2018; https://www.ema.europa.eu/en/documents/report/meeting-report-joint-biologics-working-party/quality-working-party-workshop-stakeholders-relation-prior-knowledge-its-use-regulatory-applications_en.pdf.

Corresponding author David Perez-Caballero and David Murray are principal consultants and CMC biologics experts; and Christiane Niederlaender and Kurt Brorson are technical vice presidents at Parexel International; 34-649-127-438; [email protected].