NGS-Based Assays in Cell and Gene Therapy: Enhancing Product Development

Next-generation sequencing (NGS) has revolutionized research and development in cell and gene therapy (CGT). As the number of cell and gene therapies (CGTs) continues to rise, establishing robust, reliable quality control procedures is essential to safeguard patient health and accelerate treatment delivery. 

With the rapid expansion of the CGT industry, promising therapies are emerging to address previously untreatable conditions. Patients with life-threatening diseases now have renewed hope for cures and improved quality of life. However, several challenges still limit global access to CGTs, including the difficulty of consistently ensuring critical quality attributes (CQAs) essential for therapeutic success and manufacturability. 

To achieve their intended therapeutic effect, CGTs require rigorous quality control to confirm product stability, consistency, and comparability.¹ This is especially critical for living therapeutics, which are susceptible to modifications that may compromise efficacy. 

By leveraging advanced NGS technology, biopharmaceutical and biotech companies can rapidly assess CGT product quality, minimizing patient risk and avoiding costly, reputation-damaging setbacks. 

CGTs are complex biological drug modalities, often developed from limited sample material. NGS is particularly valuable in this context, enabling comprehensive genetic analysis from minimal input. This is ideal for high-resolution characterization without compromising sample integrity.

Ensuring the Biosafety of Cell Therapies

Cell therapy development carries a higher risk of contamination by adventitious agents than gene therapy. Mammalian cells are typically cultured in media containing animal-derived components, which increases the likelihood of infection.

Biosafety must be assessed early in cell therapy development by screening for contamination in:

• Starting material collected from patients (autologous) or healthy donors (allogeneic)
• Components such as media and cytokines used to promote differentiation into specific phenotypes³

Because apheresis collections are invasive, only minimal material is collected to reduce patient burden. As a result, biosafety assessments must be conducted using limited sample volumes. Efficient testing methods are required to generate meaningful insights while minimizing waste and avoiding development delays.

Ensuring the Biosafety of Gene Therapies

Biosafety assessment of viral vectors is critical in gene therapy development. Before batch analysis, the risk of cross-contamination must be thoroughly evaluated. Regulatory authorities require data on contamination, control, and stability during the development and characterization of cells used to amplify genetic material. ⁴ Demonstrating copy number and transfection efficiency is also essential for regulatory approval of gene therapy products.⁴  

Contamination during gene therapy development may originate from media containing animal-derived material. Production cells also pose a risk, as they can introduce viruses pathogenic to humans.⁵ The Consortium on Adventitious Agent Contamination in Biomanufacturing (CAACB) at MIT’s Center for Biomedical Innovation compiled a comprehensive dataset on viral contamination incidents across all stages of the biological product lifecycle.⁵ Such contamination can lead to discarded batches, manufacturing delays, regulatory setbacks, and postponed treatment — posing serious risks to patient safety.

Genetic Characterization of CGTs for Identity and Stability Testing

Throughout CGT development, changes in conditions, materials, and methods are inevitable. As living therapeutics that respond to their environment, these therapies are susceptible to mutations that affect identity and stability, complicating the definition and maintenance of CQAs. The FDA recommends conducting identity and stability testing at least twice during development. Regular testing using characterization methods such as NGS helps monitor product integrity, ensuring consistent quality and efficacy. Genetic characterization provides essential insights to optimize stability and define appropriate shelf life and storage conditions.

The Role of NGS in CGT

NGS plays a pivotal role in CGT development and manufacturing by delivering deep insights into the genetic composition of therapeutic products. CGTs involve complex components such as plasmids, viral vectors, and engineered cells. NGS enables precise characterization of these components, confirming the presence and accuracy of genetic modifications introduced through techniques such as CRISPR. NGS-based assays verify the correct assembly of therapeutic constructs, ensuring product integrity.

Throughout manufacturing, NGS delivers essential insights into CGT product stability and identity. It detects genetic drift, unintended mutations, and structural changes that may occur during cell expansion, vector production, or storage. Monitoring product identity helps prevent adverse effects in patients, while maintaining stability throughout development and manufacturing maintains therapeutic efficacy. If stability is compromised due to contamination, degradation, or other factors, biopharmaceutical companies risk costly setbacks and non-compliance with regulatory standards.

Key Applications of NGS-Based Assays in Cell and Gene Therapy

Contamination Screening

NGS-based assays provide a sensitive, comprehensive method for detecting contaminants in cell and gene therapy products, including bacteria, fungi, mycoplasma, and adventitious viruses.¹ By sequencing all nucleic acids in a sample, NGS identifies both known and unexpected impurities, even at low abundance. To ensure optimal sensitivity, best practices in sample preparation, such as rigorous nucleic acid extraction and the use of spike-in controls, are essential. Advanced bioinformatics pipelines enhance accuracy by filtering background noise and distinguishing true contaminants from host or environmental sequences, supporting robust biosafety assessments throughout CGT development.

Viral Vector Characterization

Characterizing viral vectors during gene therapy development is essential to ensure safety, efficacy, and consistency. Because these vectors are modified viruses, they must be evaluated to confirm successful delivery of genetic material into patient cells. Incomplete, mutated, or contaminated viral vectors may compromise therapeutic function or pose safety risks. 

NGS-based assays enable validation of viral vectors used in cell and gene therapy development. These assays deliver insights into vector genome integrity, copy number, and titer, confirming that the therapeutic payload is correctly assembled and consistently delivered. Two key analytical strategies support viral vector characterization with NGS: reference-based mapping, which aligns reads to a known sequence to detect mutations or deletions, and de novo assembly, which reconstructs the genome without prior knowledge This is particularly useful for identifying unexpected variants. NGS also detects replication-competent viruses and off-target integrations, both critical for ensuring product safety. Dual-index barcodes support multiplexed workflows by enabling simultaneous tracking of multiple vectors in a single sequencing run, enhancing efficiency and scalability.

Genetic Integrity Monitoring

NGS enables precise tracking of on-target edits, insertions, or deletions (indels), and large-scale genomic rearrangements, supporting genetic fidelity monitoring in CGT. Depth-of-coverage requirements vary by therapeutic modality, such as CRISPR-edited cells, lentiviral vectors, or AAV-based therapies, to ensure accurate detection of both intended and unintended changes. Longitudinal sampling during cell expansion or storage phases helps identify genetic drift and emerging variants over time. 

Vector and Gene Editing Quality Control 

NGS-based assays verify plasmid integrity, ensuring the full sequence including complex or repetitive regions is accurate and free from unintended mutations. For viral vectors, NGS confirms the complete genome sequence, supporting consistency across production batches. In gene editing applications such as CRISPR, NGS detects off-target events, including unintended insertions, deletions, or rearrangements, which may compromise safety or efficacy. Regulatory agencies such as the FDA and EMA recommend full plasmid and vector genome sequencing, along with comprehensive off-target analysis, as part of Chemistry, Manufacturing, and Controls (CMC) submissions. These practices ensure gene therapy products meet the highest standards for precision, reproducibility, and patient safety.

Single-Cell Transcriptomics and Potency Testing

Single-cell RNA sequencing (scRNA-seq), an NGS-based application, assesses identity, heterogeneity, and potency of cell therapy products at single-cell resolution. By profiling gene expression across thousands of individual cells, scRNA-seq enables precise monitoring of cell differentiation, detection of functional markers, and identification of rare subpopulations that influence therapeutic performance. This level of granularity ensures product consistency and potency, especially in complex therapies such as stem cell-derived treatments or engineered immune cells. scRNA-seq increasingly supports regulatory expectations for robust characterization and quality control in advanced cell therapy development.

Benefits of NGS-Based Assays in CGT Product Development

High Sensitivity and Broad Detection Capabilities

Unlike traditional methods, NGS detects extremely low levels of contaminants and subtle genetic alterations that might otherwise go unnoticed. For example, NGS has detected rare viral agents such as minute virus of mice (MVM) and low-abundance bacterial contaminants such as mycoplasma in cell therapy products. These are cases where PCR or culture-based techniques lacked sufficient resolution. NGS also enables simultaneous screening for a broad spectrum of targets, including multiple microbial species and diverse genetic modifications such as off-target edits and insertional events. This comprehensive approach enhances product safety, supports regulatory compliance, and ensures the integrity of complex biological therapies. NGS-based assays offer exceptional sensitivity and broad detection capabilities, making them invaluable tools in advanced biomanufacturing and cell therapy development.

Accelerated Quality Control and Reduced Development

NGS enables rapid, high-throughput data acquisition and interpretation, significantly streamlining genomic analysis workflows. In CGT development, NGS enables real-time quality control by rapidly identifying contaminants, verifying genetic integrity, and assessing off-target effects in a single assay. This speed and multiplexing capability translates into tangible operational benefits: faster batch release through reduced turnaround times for safety and identity testing, expedited regulatory submissions supported by comprehensive datasets, and accelerated delivery of therapies to patients. By integrating NGS into early and late-stage development pipelines, organizations can reduce bottlenecks, enhance decision-making, and improve responsiveness to regulatory requirements.

Comprehensive Genomic Profiling

Whole-genome sequencing (WGS) and targeted NGS panels offer powerful tools for detecting off-target edits, vector rearrangements, and unexpected genomic integrations. These parameters are critical for the development and safety assessment of gene and cell therapies. Unlike single-target methods, which are limited to predefined loci, parallel sequencing enables simultaneous interrogation of thousands of genomic regions, providing a more complete and unbiased view of genetic alterations. This multiplexed approach enhances the detection of rare or complex events that may impact therapeutic efficacy or safety. Visual aids such as coverage maps and mutation frequency plots support interpretation by illustrating sequencing depth, variant distribution, and integration hotspots. This offers scientists and regulators a clear, data-rich view of CGT product integrity.

Enhanced Biosafety Testing

NGS is a transformative approach to biosafety testing, enabling sensitive and comprehensive detection of adventitious agents in CGT products. Unlike conventional methods such as PCR or culture-based assays, which are limited to predefined targets or require viable organisms, NGS detects a broad spectrum of known and unknown contaminants in a single, unbiased analysis. This expanded coverage significantly improves the ability to identify low-abundance or unexpected agents that may compromise product safety. Integrating NGS-based assays into quality control workflows ensures CGT products meet stringent safety standards before clinical use. By providing a more complete biosafety profile, NGS supports robust risk mitigation strategies, enhances patient safety, and facilitates regulatory compliance.

Regulatory Guidance, Acceptance, and Compliance for NGS in CGTs

The growing acceptance of NGS by international health authorities such as the FDA and EMA marks a paradigm shift in quality assurance for CGTs. Regulatory bodies now recognize NGS as a powerful tool for characterizing CQAs, detecting adventitious agents, and ensuring genetic stability. This has led to its inclusion in updated frameworks such as ICH Q5A(R2), which explicitly endorses NGS as a replacement for traditional in vivo assays. Both the FDA and EMA have issued guidance on vector sequencing requirements and biosafety testing standards, emphasizing validated analytical methods and risk-based approaches. Validated NGS assays such as viral vector genome integrity analysis, residual host cell DNA quantification, and adventitious virus detection are now routinely used to support regulatory filings, audits, and compliance documentation. These assays not only enhance data integrity and traceability but also align with GMP and GLP standards, streamlining documentation and accelerating review timelines. Strategically, this translates into faster approvals, reduced regulatory risk, and smoother market access — giving developers a competitive edge in the rapidly evolving CGT landscape.

Implementing NGS: In-House vs Outsourcing

When implementing NGS workflows for CGT quality assurance and quality control, organizations face a strategic decision: build capabilities in-house or outsource to external providers. In-house implementation offers biopharmaceutical organizations full control over their data, faster iteration cycles, and the ability to tailor assays to organization-wide and regulatory needs. However, it demands significant investment in infrastructure, bioinformatics expertise, and compliance readiness. Outsourcing provides access to validated platforms, regulatory-grade documentation, and scalability, but may limit flexibility and increase turnaround times. These trade-offs highlight the value of integrated software solutions such as those from Genedata, which bridge the gap by enabling standardized, automated, and compliant NGS data processing and analysis, whether workflows are internal, external, or hybrid. Such platforms streamline collaboration, reduce manual errors, and accelerate regulatory submissions, making them a strategic asset in the evolving CGT landscape.

Genedata Selector for Streamlined NGS-Based Biosafety and Cell Line Development Workflows

Leveraging innovative, sensitive approaches enables biopharma companies to establish robust, optimized quality control processes during CGT product development. This leads to time savings, reduced costs, and improved patient safety. Incorporating NGS into CGT development offers multiple advantages and is recommended by regulatory authorities.⁶ NGS offers broader detection capabilities than other methods, enabling targeted and untargeted adventitious agent detection (AAD).⁷ Beyond biosafety, NGS provides valuable insights into CGT products through transcriptome analysis, enabling thorough characterization of single cells or “living drugs”.⁸ Implementing NGS early in CGT development can be challenging, especially for companies without existing in-house capabilities. Designing NGS-based biosafety assays and establishing optimized bioinformatics pipelines for accurate data analysis and efficient decision-making can be complex.  

Genedata Selector for Streamlined NGS-Based Workflows

To obtain results quickly and cost-effectively, biopharma companies often outsource AAD assays to external providers offering PCR-based tests. Outsourcing requires sharing proprietary genomic data, which introduces risks related to intellectual property (IP). Additionally, PCR-based methods limit the number of detectable viruses. NGS-based assays enable comprehensive biosafety assessment without compromising speed. Implementing NGS-based biosafety assays in-house with Genedata Selector simplifies data analysis by providing optimized workflows and centralizing genetic characterization for seamless collaboration and a single chain of custody. Designed for biosafety and cell line development, Genedata Selector supports both cell and gene therapy applications.

In addition to enabling accurate risk mitigation from starting material to final product, Genedata Selector facilitates:

• Omics and RNA-Seq-based quality control of cell therapies
• Machine learning to predict cell characteristics during expansion and assess variability
• Gene editing verification, including CRISPR results, insertion sites, and plasmid stability

Built for cGMP environments, Genedata Selector centralizes sample data for transparent reporting and provides Computer System Validation (CSV) support — helping biopharma companies eliminate bottlenecks and accelerate delivery of life-changing therapies.

Learn how you can accelerate CGT product development with Genedata’s scientific experts and Genedata Selector.

Learn More about Genedata Selector

References:

1. Stroncek, D. F.; et al. Quality assessment of cellular therapies: the emerging role of molecular assays.  Korean J. Hematol. 2010, 45(1), 14–22.  
2. Cetin B et al. Gene and cell therapy of human genetic diseases: Recent advances and future directions. Journal Cellular and Molecular Medicine. 2024, 28(17)
3. Clarke, D.; Aragon, M. Optimizing the quality of cell therapy starting materials. RegMedNet. 2018. https://www.regmednet.com
4. European Medicines Agency (EMA). Guidelines on the quality, non-clinical and clinical aspects of gene therapy medicinal products. 2018.
5. Barone, P. W.; et al. Viral contamination in biologic manufacture and implications for emerging therapies. Nat. Biotechnol. 2020, 38(5), 563–572.
6. World Health Organization (WHO). Proposed 1st International Virus Reference Standards for Adventitious Virus Detection in Biological Products by Next-Generation Sequencing (NGS) Technologies (CBER-5). 2020.
7. Charlebois, R.L.; et al. Sensitivity and breadth of detection of high-throughput sequencing for adventitious virus detection. npj Vaccines 2020, 5, 61.
8. Tzani, I.; et al. Tracing production instability in a clonally derived CHO cell line using single-cell transcriptomics. Biotechnol. Bioeng. 2021, 118(5), 2016–2030.