Next-Gen Sequencing for Adventitious Virus Testing

This highly effective testing method removes animal testing from product release requirements, a 3Rs plus

For decades, vaccine developers relied on animals to test whether their products contained adventitious viruses-the scientific term for viruses that unintentionally get introduced into the manufacturing process. Laboratories historically used a combination of in vivo, in vitro and PCR-a tool that amplifies a segment of DNA-to track these unwanted microorganisms.

But a new tool is stepping up and taking its place in a big way. Innovative next-generation sequencing (NGS) approaches have been developed that can detect live viral contaminants in cells, and regulatory bodies are encouraging its use as a replacement for in vivo assays. Two of Charles River's Biologics experts, Olaf Stamm, PhD, Technical Business Development Director and Horst Ruppach, PhD, Scientific and Portfolio Executive Director with Global Biologics, answer some of the frequently asked questions that clients have about the use of NGS as an adventitious virus assay, from its utility in the lab to what regulators are saying about it.

With recent developments in NGS-based viral safety testing is in vivo adventitious agent (AVA) testing still the best way forward?

Certainly not. NGS-based testing overcomes the limitations of traditional in vivo methods, such as interference, the need for neutralization of the (product) virus, and the risk of false negative and false positive results. Traditional virus assays were developed and used simply because there were no better alternatives available at the time. In fact, in vivo assays have been shown to be inferior to in vitro assays; contamination events detected in cell-based assays were sometimes missed in parallel in vivo tests. This discrepancy is likely because animals have immune systems, whereas cells do not.

With the advent of powerful NGS-based solutions, such outdated approaches should be discontinued immediately, both for scientific and ethical reasons.

Why do you feel internal teams should look at NGS testing as a potential alternative to in vivo AVA methods?

Historically, virus testing relied on a combination of different assays with orthogonal principles, as no single assay could fully address the complexity of viruses. Observing animals for symptoms of viral infection after administering the product of interest was a logical approach in the past, but it is no longer justifiable. With the advent of NGS, we now have a powerful, unbiased tool that is unaffected by product or matrix interference, agnostic-meaning it can detect all viruses-and is also highly sensitive.

Which assays of cell line characterization packages other than in vivo AVA can be replaced by NGS in the future?

In consideration of the power of NGS, in principle almost all non-functional assays could be replaced. This includes the antibody productions tests (MAP/HAP/RAP), dedicated virus panels such as the 9CRF tests for bovine and porcine derived viruses as well specific virus tests such as minute virus of mice (MVM) or Calicivirus on CHO cells. For human cell lines the agnostic NGS test would omit the need for testing the panels of human/simian viruses because no PCR panel will be as comprehensive as the agnostic NGS transcriptomic approach.

Looking at regulatory approvals, what is the best strategy today to implement NGS-based testing methods while being in the transition phase?

The use of NGS to streamline the cell line characterization process requires a shift in mindset among manufacturers and regulators. For decades, the industry has seen QC testing become increasingly comprehensive and complex. However, NGS reverses this paradigm by replacing a suite of assays with a single, more effective method. The recent revision of the ICH Q5A guideline demonstrates regulators' readiness to embrace modern techniques like NGS. A strategic approach today would involve replacing all in vivo assays and specific virus tests/panels while retaining functional assays such as in vitro AVA, cell-based retroviral testing (e.g., S+L−), and electron microscopy. This approach significantly enhances safety, reduces time and costs, and is likely to gain acceptance from any regulatory body involved in the ICH process.

How is this testing impacting EU vs US vs APAC markets?

With the 3Rs initiatives (replace, reduce, refine the use of research animals) evident in both the EU and the US, these markets have an additional incentive to move away from traditional in vivo testing models. Although explicit programs like these are not yet prevalent in APAC, the other advantages of NGS-based AVA testing are convincing on their own.

There are no scientific or regulatory arguments that justify the continued use of traditional assays. However, it must be acknowledged that quality control is typically not an area known for innovation or early adoption of new technologies. Therefore, it is the responsibility of CROs to provide comprehensive and compelling data packages that clearly demonstrate the benefits of the NGS approach.

Well-presented data on performance (coverage, specificity, and sensitivity) and compliance (method validation and instrument qualification) will help address concerns and ultimately persuade clients and regulatory bodies to adopt these new technologies and this will include the APAC market.

What are the advantages of NGS vs. Animal Testing

NGS provides timely and sustainable results and completely removes animal testing from your product release requirements. A major advantage of NGS has been highlighted by recent regulatory changes driving the reduction in the use of animals for viral safety tests. To comply with the 3Rs principles on animal experimentation, the regulatory bodies are indeed pushing to limit the use of animal models only when there are no other alternatives.

The ICH's revision 2 (R2) highlights the benefits of Next Generation Sequencing assays versus in vivo assays: 3.2.3: "NGS is encouraged as a replacement for in vivo assays because it can overcome the limitations of the breadth and sensitivity of virus detection of the in vivo assays. Furthermore, this promotes the global objective to replace, reduce, and refine the use of animal testing."

By the way, the guideline also refers to NGS as a replacement for Mouse, Rat, and Hamster Antibody Tests (3.2.4).

Lastly, what are some specific benefits of using NGS testing?

There are many. Very low sample volumes are needed for testing and faster turnaround times allow you to meet tight clinical timelines. You also have the advantage of robust and powerful bioinformatics - including an automated BioIT pipeline, and the ability to identify a broad range of viruses, including still unknown viruses. Other benefits of NGS testing include:

• The opportunity to replace multiple standard virus tests with one NGS assay
• Discriminating infectious virus from non-replicating carryover or dormant virus
• More efficient and comprehensive genetic characterization of production cell banks and gene therapy products
• Faster drug development due to faster, comprehensive safety testing

NGS represents a superior approach for the detection of adventitious viruses. It shares PCR's insensitivity to potential drug toxicity and goes beyond this by sensitively detecting both known and unknown or variant viruses. This makes NGS an optimal virus screening and virus characterization strategy, particularly when speed, sample volume, and the challenge of antibody neutralization are pressing concerns.

Minu Youngkin handles global partnerships and strategic partnerships for Charles River's Biologics business.

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