Next Generation Sequencing in Adventitious Agent Testing

Companies developing allogeneic cell therapy products (derived from donors or stem cells/iPSC) have recognized the importance of the NGS transcriptomic approach to demonstrate viral safety of the final cell therapy product. The conventional tools will fail to detect certain viruses because of the inappropriateness of some methods to detect certain human or zoonotic animal viruses. For instance, the cell-based in vitro AVA assays use immortalized cell lines to detect viruses.

It is well known that many primary viral isolates (directly isolated from humans) do not grow in immortalized cell lines. Examples include Hepatitis C, Hepatitis E, different herpes viruses (HHV6/7), and many HIV variants. The in vivo assays, including the MAP/HAP/RAP assay, can detect viruses infecting humans but are limited to those that grow in rodents. The typical human virus PCR panel will detect those viruses, but variants of the same might be missed. The agnostic virus screen by the NGS-based transcriptome assay will provide the highest assurance of viral contaminant absence.

There may be virus-related reads that remain after the filtering and verification steps in the bioinformatics pipeline. These are assessed for relevance as part of the service, with expert interpretation included in the final NGS report to explain the rationale behind any unidentified hits. In most cases, such reads can be invalidated as false positives. Proprietary algorithms are designed to filter out non-relevant sequences or, in the case of a true hit, identify the virus. Even novel or previously uncharacterized viruses often show similarity to known viral sequences in the database. An AI-based validation tool is in development to support this process further, drawing on data from analyses performed over the past 5 to 6 years and continuing to expand with each new dataset.

In principle, NGS (transcriptome approach) applied on cells isolated from the donor would be suitable to support donor selection and presumably be superior from a quality and safety perspective. It would perfectly supplement the current donor testing performed at the donation centers using PCR and other less sophisticated methods typically applied on blood samples (not cells). Turnaround time and cost might be a challenge today, and a multiplex PCR can be an alternative solution, but as the technology scales up, the situation might change. The benefit of the agnostic NGS transcriptome approach is that it will not miss any virus, unlike a multiplex PCR.

For a valid answer, we would need more context about this project, product, or test item submitted to NGS. For vaccines, the FDA has been pushing manufacturers for years to use NGS, while for recombinant protein products, the agency became open to it with the ICH Q5A(R2) guidance released in November 2023.

The number of submissions including NGS solutions is increasing yearly (most recently reported by the FDA at the IABS conference in Frankfurt, December 2024), which will lead to higher acceptance. As outlined in our presentation, agencies request many details, and we have submitted the whole validation package and associated data via the Master File approach at the FDA.

It's also important to note the draft EP 2.6.41 provides details on method controls and method validation, which will also support NGS acceptance. The document passed the comment phase last spring and is expected to be published mid-year.

Yes, with the most recent adoption of NGS by the ICH guideline, all countries have formal regulatory guidance coverage for this technology. However, regulators in different countries have a different level of knowledge about new technologies, and therefore, some regulators might raise questions. Typically, some agencies in Asia have shown to be more conservative, although they are even part of the ICH process. Finally, any regulatory decision depends on the information provided to the assessor and his/her confidence about a new technology. At Charles River and PathoQuest, we actively support clients with appropriate documentation and feedback in case of questions.

There are cases where an NGS approach is more reasonable than the in vitro AVA test. Testing master virus seeds or attenuated viral vaccines by NGS (viromic approach) will be accepted because of the test item interference in the in vitro AVA tests and the low value when analyzing control cells only.

In principle, NGS can replace in vitro AVA testing, too. However, since some regulators still need to get used to this new approach, it might be helpful to keep the in vitro assay for cell line characterization programs. This perception might change in the future once all regulators have experienced NGS and begin to recognize it as a powerful virus screening tool. The data supporting this approach is increasing, and we have demonstrated the power of the NGS approach to detect a broad range of viruses.

The regulatory expectations on NGS validation are well summarized in the draft EP 2.6.41; it is worth noting that representatives of the FDA were involved in drafting this EP document. There is also a great industry white paper providing many details on this topic. The general principle is to validate critical steps individually (e.g., nucleic acid extraction, library preparation, etc.) in addition to a validation covering the whole process from sample pre-treatment to data processing via the bioinformatics pipeline.

Spiking experiments are an integral part of some NGS approaches, such as viromics, which directly target viruses. For these approaches, reference virus strains are available through the WHO.

The benefit of the NGS transcriptome approach is that it addresses viral mRNA, which is a high indicator of infectious viruses. Residual viral genome nucleic acids from DNA viruses won’t go into the analytics. Residual viral RNA genomes can also be differentiated from the viral mRNA; the +/- sense strand information is retained in the sequencing approach (for more details see A. Brussel, et. al. Use of a new RNA next generation sequencing approach for the specific detection of virus infection in cells; Biologicals, Volume 59, May 2019, pages 29-36). Of course, genome coverage and depth of reads (amount of reads) per RNA segments are critical aspects to assess the findings. The mRNA signature can also be an indicator for lysogenic viruses.

In principle, NGS can replace the in vitro AVA testing, too. However, since some regulators still need to get used to this new approach, it might be helpful to keep the in vitro assay for cell line characterization programs. This perception might change in the future once all regulators have experienced NGS a the most powerful virus screening tool. The data supporting this approach is increasing, and we have demonstrated the power of the NGS approach to detect a broad range of viruses (see video). There are cases where an NGS approach is more reasonable than the in vitro AVA test. Testing master virus seeds or attenuated viral vaccines by NGS (viromic approach) will be accepted because of the test item interference in the in vitro AVA tests and the low value when analyzing control cells only.

Absolutely, yes. The vaccine industry was the first community to embrace the NGS technology (Viromic approach) for contamination testing of viral seed stocks, especially since NGS omits the need for neutralizing antibodies or the tedious testing of the control cells. Vaccine companies have successfully submitted NGS data as a replacement of in vitro AVA for master virus seed or attenuated viral vaccine crude harvests.