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Advanced NGS Techniques for Cell Line Characterization in Cell and Gene Therapy

April 21, 2025
Valentina Armiento

Cell and gene therapies provide efficient treatments for a variety of medical conditions1. Central to the success and safety of these therapies is detailed characterization of the cell lines, which involves ensuring genetic stability, phenotypic consistency, and absence of contaminants. Given that most of these therapeutics are produced using living cells, meticulously characterizing the cell lines and assessing the quality of raw materials is critical. Such a stringent approach mitigates any potential adverse effects on the cell lines and the overall manufacturing process. Standardized regulations and next-generation sequencing (NGS) enable comprehensive characterization and rigorous quality control, ultimately reducing time-to-market. 

Regulatory bodies, such as the FDA and the EMA, have provided harmonized guidelines for monitoring critical quality attributes (CQAs) of cell and gene therapies. The Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) define a CQA as “a physical, chemical, biological or microbiological property or characteristic that should be within an appropriate limit, range, or distribution to ensure the desired product quality.”2 Due to its increased access and affordability, NGS can be applied to monitor genetic modifications, detect off-target effects, and ensure the overall efficacy and safety of cell and gene therapies. NGS offers comprehensive detection capabilities, which can identify potential issues or contaminations from early-stage development all the way to manufacturing. 

However, NGS assays generate large amounts of complex data, which require careful management and analysis. This complexity is compounded by the need to validate multiple NGS assays, making their integration into existing frameworks and workflows challenging. Data bottlenecks and silos can further hinder the smooth flow and accessibility of information needed for thorough analysis. By bringing the NGS assays in-house, these bottlenecks can be better controlled or even eliminated, thereby reducing internal business risk. Therefore, using a specialized software solution to address in-house NGS assay challenges has become essential for comprehensive cell line characterization and quality control. 

Characterization of Critical Quality Attributes in Advanced Therapies

NGS offers several key applications in the characterization of CQAs of cell lines used for CGTs. It is frequently used for copy number analysis in producer and packaging cell line manufacturing platforms. By analyzing the copy number of integrated vectors, researchers can select cell lines with optimal gene integrations, thus ensuring genetic stability and consistency. NGS is also applied in cell line identification, specifically for detecting on-target and off-target modifications, which is crucial for verifying the accuracy of genetic modifications. Additionally, it facilitates integration site analysis for lentiviral vectors (LVV) and CRISPR/Cas-9 gene editing results, providing comprehensive insights into the genomic integration of therapeutic vectors. 

Ensuring reproducibility and thorough product testing before release must also be addressed. The characterization process starts with identity testing to confirm the intended cellular components and quantify any extraneous safety concerns. Most cell therapies include a purification step to eliminate potential adventitious agents and contaminants. Furthermore, potency is evaluated as a key CQA to ensure the product retains the necessary biological function for its intended clinical use3.

Gene therapies deliver genetic material via vectors, such as viruses, to treat or prevent diseases. Their identity is defined by the genetic material and its delivery system. Verifying the integrity, potency, and safety of the product remains essential. The genetic material within the vector must be intact and correctly sequenced for the therapy to be effective. Assessing the potency of the gene therapy ensures it can deliver the genetic material to target cells and achieve the desired therapeutic effect. Rigorous testing confirms the absence of replication-competent viruses and other contaminants, ensuring the therapy is safe for patient use.

NGS-assays supported by the Genedata Selector measure Critical Quality Attributes of of cell and gene therapies, such as identity, integrity and safety.

Mitigating Risks in CGT Production

Assessing and mitigating risks associated with cell lines and starting materials in CGT production is critical for timely therapy delivery. The complexity of cell and gene therapies, along with the lack of standardized requirements for clinical-grade starting materials, presents challenges for companies developing and manufacturing these products. The inherent variability of raw materials, including excipients, reagents, chemical additives, source material, adventitious vectors, and processing components4, can significantly impact the potency and safety of the final product.

The risk assessment process involves identifying and mitigating risks early in development to prevent delays in clinical trials and commercialization. This includes verifying cell line origin, genetic stability, and absence of contaminants, along with regular testing for mycoplasma and viruses. Implementing standardized protocols for cell line identification and characterization helps mitigate risks associated with genetic drift and variability. Since CGTs are produced in living cells, adherence to regulations such as the FDA’s guidelines on human- and animal-derived materials5 is vital for ensuring product safety and efficacy.

Leveraging the Advantages of NGS In-House 

Robust in-house NGS data processing and management are essential to fully harness the potential of NGS in CGT development. Utilizing in-house NGS assays offers numerous benefits, including a lower cost per sample, potential for quicker turnaround times, and the ability to protect intellectual property. Genedata Selector® was developed to support end-to-end in-house NGS workflows in biopharmaceutical organizations and to streamline NGS data into meaningful scientific insights. This comprehensive, off-the-shelf analysis system is designed for all NGS-based QC assays, simplifying sample management and data analysis during CGT development and manufacturing. It breaks down data silos, tracks assay parameters and reference sequences to ensure reproducibility, and accommodates diverse CGT workflows. Powered by Playbooks, wizard-based guides that make NGS analytics easier to follow, the system facilitates a straightforward assessment of multiple CQAs within a single assay. 

Genedata Selector can be validated in a good manufacturing practice (GMP) environment within a company's own facilities, streamlining the NGS assay validation process across multiple modalities. This approach is more efficient compared to independent assay validation, which can be complex and fragmented. It automates NGS data analysis workflows and generates comprehensive reports for internal evaluation and regulatory submissions. Genedata’s quality assurance expertise ensures all processes meet the highest standards of quality and safety. Its framework for NGS compliance in GMP enables organizations to continuously add and validate new NGS assays, expanding their testing repertoire to new assay types and modalities on a single, central NGS analysis platform. 

In conclusion, the integration of advanced NGS techniques with an organization’s existing infrastructure is essential for the detailed characterization and quality control of cell lines in cell and gene therapy development. By addressing the complexities of data management and ensuring the thorough assessment of critical quality attributes, biopharmaceutical organizations can enhance the safety and efficacy of their therapies. Software such as Genedata Selector streamlines these in-house processes, breaking down data silos and supporting diverse workflows. Embracing a specialized software solution to address in-house NGS assay challenges not only mitigates risks but also accelerates the development of innovative treatments. 

 

References

  1. Roy, R. et al.Cell and Gene Therapy: Current Landscape, Emerging Trends and Future Prospects.
  2. ICH. Committee for Human Medicinal Products ICH Guideline Q8 (R2) on Pharmaceutical Development. www.ema.europa.eu/contact (2017).
  3. Karanu, F., Ott, L., Webster, D. A. & Stehno-Bittel, L. Improved harmonization of critical characterization assays across cell therapies. Regenerative Medicine vol. 15 1661–1678 Preprint at doi.org/10.2217/rme-2020-0003 (2020).
  4. Scott, M. et al. Transitioning from development to commercial: risk-based guidance for critical materials management in cell therapies. Cytotherapy22, 669–676 (2020).
  5. FDA. Considerations for the Use of Human-and Animal-Derived Materials in the Manufacture of Cellular and Gene Therapy and Tissue-Engineered Medical Products Draft Guidance for Industry. www.regulations.gov. (2024).