Bringing Next-Gen Sequencing To GMP Scale

A conversation between Jihye Nam and Tracie Fradet at Astellas Pharma and Life Science Connect's Jon O'Connell

Detecting unwanted virus particles in cell and gene therapies, which are complex and often provide limited sample volumes, raises specific challenges for traditional detection tools. Conventional assays can be slow and resource-intensive. Beyond that, you need to know what viruses you're looking for before you even begin the assay.

Next-generation sequencing is attracting attention as a broader, more information-rich approach to adventitious agent detection. NGS can scan for a wide range of viral signals in a single assay, creating new possibilities for GMP-scale viral safety testing. On the other hand, it raises new questions about validation, interpretation, and regulatory alignment.

Jihye Nam and Tracie Fradet of Astellas Pharma recently gave us a preview of their upcoming talk at the 2026 ISPE Annual Meeting & Expo. They discuss why NGS is gaining traction for cell therapy manufacturing, how early conversations with regulators can shape implementation strategy, and what scientific and operational challenges remain as the industry works to bring this technology into routine GMP use.

For readers less familiar with virus detection methods, what are the limitations of traditional assays that make NGS necessary or preferred?

Traditional adventitious agent detection assays have played a critical role in ensuring the safety of biopharmaceutical products, but they have several inherent limitations. Cell culture-based assays depend on a virus' ability to infect specific indicator cells and produce visible effects, which can require up to 28 days of observation and may be impacted by sample-related interference. In vivo assays similarly rely on viral replication and observable pathology, have variable and often poorly defined sensitivity, require lengthy testing periods, and involve more testing — these conflict with the industry's commitment to the 3Rs principle of replacement, reduction, and refinement. Molecular assays offer faster results but are limited to the detection of known viruses for which specific sequence targets have been designed.

Next-generation sequencing (NGS) addresses many of these challenges by providing a hypothesis-free approach that does not require prior knowledge of viral sequences. NGS enables broad detection of both known and novel viruses in a single assay, while offering high sensitivity, specificity, and scalability. As a result, NGS can complement, supplement, or potentially replace conventional assays, improving efficiency while strengthening viral safety assurance for GMP-manufactured cell therapy products.

Does adventitious agent detection carry different stakes for cell and gene therapy compared to traditional biologic modalities?

While adventitious agent detection is critical for all biopharmaceutical products, cell and gene therapies present unique challenges that can increase the complexity of viral safety testing. Unlike many traditional biologics, cell therapy products contain large amounts of host cell nucleic acids. This significant host background can reduce assay sensitivity by masking low-level viral signals, making robust sample preparation and bioinformatic analysis essential.

Cell and gene therapy products can also be constrained by limited sample availability, especially when testing valuable cell banks, intermediates, and drug products. Unlike traditional biologics, where larger sample volumes may be available for release testing, every sample consumed during testing may represent a meaningful portion of a finite manufacturing lot. In this context, information-rich technologies such as NGS provide significant value because they can generate an extensive amount of safety data from a small sample input. The ability to interrogate a broad range of known and potentially novel viral agents within a single assay reduces the need for multiple orthogonal tests and maximizes the amount of safety information obtained from limited test material. This efficiency is particularly important for advanced therapy products, where minimizing sample consumption while maintaining a comprehensive safety assessment is a key consideration.

In addition, cell and gene therapy manufacturing processes often use complex raw materials, including cytokines, plasmids, and viral vectors. Residual nucleic acids from these materials may generate false positive signals during testing and must be carefully evaluated in the context of the sample matrix and manufacturing process. Consequently, detecting a viral sequence does not always equate to the presence of an infectious contaminant. For NGS-based adventitious agent detection, distinguishing true viral contamination from background sequences is a critical component of assay interpretation. These considerations underscore the importance of early regulatory alignment, rigorous method development, and risk-based data evaluation when implementing NGS for GMP manufacturing.

You mention industry consortia as a mechanism for aligning on best practices. Can you point to a specific scenario in which consortium alignment changed how your team approached NGS implementation?

One example is the industry's collective emphasis on early regulatory engagement when implementing NGS for adventitious agent detection. Through interactions with consortia and cross-industry working groups, we observed growing alignment around developing and validating the method first, then seeking regulatory feedback before incorporating it into GMP product testing strategies. Following this approach, our teams engaged the FDA through a Type D meeting to discuss method development and validation to obtain agency feedback prior to implementation in product testing plans.

Consortium activities have also helped promote harmonization through the use of common resources, including viral reference materials such as the First World Health Organization International Reference Panel for Adventitious Virus Detection and the Reference Viral Database (RVDB). These tools provide a shared foundation for assessing assay performance, establishing validation expectations, and standardizing bioinformatic workflows. As a result, sponsors can generate more comparable data sets and report results using a more consistent framework, which helps facilitate regulatory review and advances broader industry adoption of NGS-based adventitious agent detection.

In the context of early engagement with regulators, how far ahead of a submission are you initiating conversations? Perhaps more importantly, what are you asking regulators to weigh in on at that stage?

We recommend engaging regulators as soon as a method has been sufficiently developed and an initial internal validation package is available to support a meaningful technical discussion, but before the method is fully implemented in GMP product testing. This allows sponsors to present substantive method performance data, obtain agency feedback on the proposed use and validation package, and, where appropriate, update the method and amend or supplement the validation before final GMP implementation.

At this stage, we seek input on the comprehensiveness of the entire workflow, including sample pretreatment, nucleic acid extraction, sequencing strategy, the use of in-process controls, and the overall validation approach. Regulators are also highly interested in the bioinformatics component of NGS-based testing, so sponsors should be prepared to discuss the software platform, computational workflow, sequence classification methods, reference databases, and measures used to ensure accuracy and reproducibility of the analysis.

Another important topic is the follow-up strategy for positive findings. We discuss how viral sequence detections will be investigated, confirmed, and assessed for product impact, recognizing that not every detected sequence represents an infectious contaminant. It is also valuable to address how the strategy will manage novel or poorly characterized viruses whose genomes may not be represented in reference databases such as RVDB. Establishing alignment on these topics early can help minimize regulatory uncertainty and support successful implementation of NGS for GMP manufacturing.

Are FDA and other agencies asking fundamentally similar questions about NGS-based AAD, or have you encountered different expectations across jurisdictions?

FDA, PMDA, and other major agencies are generally asking similar scientific questions regarding NGS-based adventitious agent detection. Across jurisdictions, regulators focus on validation strategy, analytical sensitivity, breadth of virus detection, bioinformatics controls, and interpretation of positive findings. However, an important distinction is whether NGS is being proposed to supplement existing adventitious agent testing or to replace a traditional assay. When used as part of a broader virus safety strategy, regulatory discussions tend to focus on how NGS adds value and complements existing controls. In contrast, when NGS is proposed as a direct replacement for a conventional assay, regulators expect evidence that it can meet the specific performance objectives of the assay being replaced. Recent PMDA guidance discusses the use of NGS assays as potential replacements for certain conventional adventitious virus tests, whereas complete replacement of traditional in vitro adventitious virus testing is still considered premature pending greater international consensus on validation approaches.

Another area of increasing alignment is the expectation that validation should address the comprehensiveness of the entire NGS workflow rather than sequencing performance alone, as we just explained earlier. Ph. Eur. 2.6.41 and PMDA’s recent Early Consideration document both support an end-to-end, fit-for-purpose approach. PMDA further emphasizes tailoring the evidence package to the performance objectives of the assay being replaced, rather than applying a single universal validation paradigm.

FDA's public position has generally emphasized a similarly fit-for-purpose, risk-based evaluation supported by robust validation, although its public communications have tended to focus less on defining specific replacement boundaries and more on demonstrating adequate performance and scientific justification for the proposed use case. Overall, the agencies appear aligned in their direction of travel under the framework established by ICH Q5A(R2), with differences today being more about the maturity of evidence needed for particular replacement claims than about acceptance of NGS technology itself.

Validation expectations for a method like NGS are still evolving industrywide. What's your view on validating an unbiased detection method that isn't looking for one specific known target? In other words, how do you validate for the unknown?

Validating an unbiased NGS method requires a shift from validating detection of a single predefined target to validating the overall capability of the method to detect a broad range of viral agents. A critical element is the use of a comprehensive virus panel in the method validation that represents diverse virus families with different physicochemical and genomic properties, including DNA and RNA viruses, varying genome sizes, enveloped and non-enveloped virions, and different genome structures. This allows the assay to be challenged across a representative range of potential adventitious viruses and supports evaluation of sensitivity, specificity, repeatability, and robustness for broad virus detection. Ultimately, the goal is to validate the system's ability to detect a broad spectrum of viruses and to identify unexpected viral signals that warrant further investigation. In this sense, validation focuses on demonstrating the sensitivity, breadth, and robustness of the overall detection framework, providing confidence that known, divergent, and potentially novel viral contaminants will be identified and appropriately evaluated.

Equally important is the use of a comprehensive and well-curated reference database of viral sequences. The RVDB, developed by a group at the FDA's CBER, provides a harmonized resource containing sequences from known viruses and potential variants. For emerging or novel viruses, it is important to understand the limitations of the bioinformatics pipeline. Sequence divergence from database entries, partial homology to known viruses, or entirely novel viral genomes may not always result in direct identification, but the bioinformatics workflow should still be capable of flagging abnormal findings, such as low alignment rates or assembled sequences requiring follow-up investigation.

Can you talk about the tangible timeline and development benefits of implementing NGS compared to traditional assays, assuming the developer is aligned with regulators on intended use, validation expectations, and control strategy?

For our cell therapy program, implementation of an NGS-based adventitious agent detection platform required approximately 12 to 15 months of development, including assay optimization, qualification/validation activities, and installation and validation of the bioinformatics software and analysis pipeline. The timeline can vary considerably depending on the availability of resources and the effort required to validate computational systems within a GMP environment.

Once implemented, however, the benefits can be substantial. Traditional virus safety testing requires expansion and subculture of cell banks to generate sufficient material for the multiple in vitro, in vivo, and molecular assays needed for product release. This consumes valuable manufacturing personnel time, cleanroom capacity, and laboratory resources. An NGS-based approach can significantly reduce this burden by consolidating broad viral screening into a single assay.

Another key advantage is that NGS is agnostic to the identity of the virus being sought. Rather than testing specific known agents, the method can detect a much broader range of known and potentially novel viruses from a single analysis, without prior knowledge of their sequence. In addition, once the sequencing platform, bioinformatics pipeline, and quality systems have been established and validated, expansion to new products, modalities, or applications becomes much more streamlined.

Looking ahead, the potential for NGS in cell and gene therapy extends well beyond adventitious agent detection. For genetically engineered cell therapies in particular, NGS can serve as a multi-attribute method, enabling multiple product quality and safety questions to be addressed from a single sequencing run. This creates opportunities to improve efficiency, increase process understanding, and reduce overall testing complexity as the field continues to mature.

Say you establish strong regulatory alignment — what challenges (scientific, cost, etc.) remain unresolved?

Even with strong regulatory alignment, several practical and scientific challenges remain. As cell and gene therapy programs advance from development into commercialization, the resources required to maintain a GMP-compliant NGS program increase significantly. This includes ongoing management of validated software systems, data storage infrastructure, cybersecurity controls, change management, and periodic review of bioinformatics databases and workflows.

Technology also continues to evolve. Sequencing platforms, library preparation reagents, software tools, and computational infrastructure inevitably require replacement or upgrading over time. Each change must be carefully assessed and may require bridging studies, requalification, or revalidation activities to demonstrate that assay performance remains unchanged.

From an operational perspective, NGS-based testing requires a multidisciplinary skillset spanning molecular biology, sequencing technologies, bioinformatics, and data interpretation, as well as knowledge of regulatory expectations. Building and maintaining a sufficient pool of trained and qualified personnel capable of executing and reviewing these complex workflows in a highly regulated environment remains an ongoing challenge as testing demand increases.

Scientifically, one of the most important unresolved areas is the management of positive findings. NGS is highly sensitive and can detect low-level viral sequences from a variety of sources, including raw materials, environmental contaminants, or process-related background. As adoption expands, organizations need to align internally on robust risk-based follow-up strategies that define how positive signals are investigated, confirmed, interpreted, and ultimately linked to product quality and patient safety. Beyond adventitious virus detection, broader NGS applications such as analysis of pathogenic genetic variants introduce additional challenges. Distinguishing biologically meaningful findings from benign background signals can be complex, particularly when variants may impact product functionality, stability, or manufacturing performance but are not formally classified as pathogenic or associated with oncogenic outcomes. Developing scientifically sound frameworks for interpreting these signals and making consistent product quality decisions remains an important area of ongoing industry evolution.

About The Experts:

Jihye Nam is a CMC regulatory affairs lead at Astellas Pharma. Previously, she worked in GMP compliance and quality assurance for Bayer and Celltrion, respectively. She received her Master of Engineering degree from University of Illinois Urbana-Champaign.

Tracie Fradet is an associate director of cytogenomics at Astellas Pharma where she leads genetic stability assessments. Past roles include senior assay development and quality control. She received her Master of Science in regulatory affairs from Northeastern University.