Evaluation of next generation sequencing (NGS) as a rapid, alternative assay for in vivo and in vitro adventitious virus testing for vaccine safety

This project proposes to: 1) compare NGS with currently used in vivo animal assay and in vitro cell culture and PCR assays; and 2) develop a well-qualified reference virus database (RVDB II)

Industry Need

Next generation sequencing (NGS) is a powerful tool to broadly and rapidly detect adventitious viruses for enhancing safety and product development of vaccines and other biologics.
While ongoing efforts have demonstrated feasibility for standardization and validation of this complex technology for adventitious virus detection, the industry could benefit from enhanced confidence in the use of NGS.

Approach

The FDA, along with GSK, MilliporeSigma, and the University of Delaware, will:

Compare NGS with currently used in vivo animal assay and in vitro cell culture and PCR assays to determine the applications of NGS as alternative assays
Develop a well-qualified reference virus database (RVDB II) that will facilitate specific detection of viruses for accurate interpretation of NGS data for adventitious virus detection.

Impacts

Reduction in the use of animals for adventitious agent testing

Streamline and standardize bioinformatic analysis pipelines

Decrease cost and time required for adventitious virus testing, helping to facilitate more rapid product development

Assist regulatory agencies with decision-making and policy development regarding the use of NGS for replacing the in vivo animal assays and for replacing or supplementing the in vitro assays currently used for virus testing

Value Statement/Outcomes

This project demonstrated an NGS‑based safety assay that reduces reliance on animal testing for drug companies, resulting in risk benefits across vaccine safety testing.

Outputs/Deliverables

NGS study results supported replacement of in vivo animal assays and supplementing or replacing the in vitro cell culture assays.

Automated script was developed for updating the Reference Virus Database.

User friendly website features were implemented Annotation pipeline was developed

Publications

Chin, P., Tsou, J., Armstrong, A., Deneyer, N., Zanda, V., Ayama-Canden, S., Colinet, A., Fuentes, S., Korokhov, N., Lavorgna, L., Noll, M., Olgiati, S., Protz, M., Shahjahan Shaid,S., Sohrabi, A., Whiteman, M., & Arifa S. Khan, (2025) Evaluation of high-throughput sequencing for replacing the conventional adventitious virus detection assays used for biologics, https://doi.org/10.1038/ s41541-025-01351-2

Posters

Khan, A. S., Tsou, J., Chin, P., Armstrong, A., Shaid, S., Polson, S. W., & Deneyer, N., PC3.1-305: Evaluating capabilities of NGS for adventitious virus detection as an alternative method for in vivo animal assays, in vitro cell culture assays and PCR assays, NIIMBL National Meeting, Washington, DC, June 27, 2023.

Presentations

Khan, A. S., Polson, S. W., Armstrong, A., & Shaid, S., Evaluating capabilities of NGS for adventitious virus detection as an alternative method for in vivo animal assays, in vitro cell culture assay, and PCR assays, 2023 PDA Virus, Madrid, Spain, June 20, 2023.

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Project Lead

Federal Stakeholder: Food and Drug Administration

Participating Organizations

EMD Millipore Corporation

GlaxoSmithKline, LLC

University of Delaware