Inherently vulnerable to viral contamination, biologics rely on animal- or human-derived starting materials and complex manufacturing processes. Albeit rare, viral contamination incidents are dangerous and disruptive. And, as product complexity and moiety diversity increase, accompanied by demands for accelerated production, the limits of traditional viral detection methods are stressed.
Next-generation sequencing (NGS), which enables fast, agnostic, broad, and highly sensitive virus detection and identification, is an alternative to entrenched traditional methods. A notable point of reference is the 2023 revision of ICH Q5A, the lead guideline for viral safety testing. The guideline, along with other regulatory documents, emphasizes the strengths of NGS and makes recommendations for its use.
A range of in vivo and in vitro traditional methods exist, varying from animal adventitious virus assays, antibody production assays in mice (MAP), hamsters (HAP), and rats (RAP), cell-based assays, 9CFR tests targeting predefined virus panels in select species, PCR, and electron microscopy (EM).
Viral clearance during the production process also can be extremely effective but is only applicable for products like cell-derived recombinants and antibodies with extensive downstream purification processes.
No one method is perfect. In fact, often, several need to be deployed such as in cell bank testing, and still novel or greatly mutated viruses may remain undetected. As an example, 9CFR tests, while useful for known pathogens, fail to detect other species-specific viruses including new, emerging, or uncharacterized viruses.
Furthermore, animals must be susceptible to the virus in question, and cell-based assays only detect viruses that can replicate in the indicator cells and induce phenotypic changes of the cells and/or cell death and/or adsorb/agglutinate erythrocytes.
Many of these traditional methods take weeks, some up to six, to accomplish and, when positive hits result, the identity of the viral containment is typically not disclosed.
NGS expands the scope
Although many companies with established viral testing protocols are reluctant to change, increased testing speed and lower costs are enticing. A new approach is clearly desirable in the growing biologics market according to Horst Ruppach, PhD, executive director scientific and portfolio at Charles River Laboratories.
“Simply use NGS but maybe not initially in a critical submission. In new product development use it as an alternative or a supplement to the standard testing to get familiar with the technology and to gain experience. We can help facilitate a plan to understand the technology,” advised Ruppach.
NGS, without bias, detects, sequences, and identifies all nucleic acid molecules within a sample. Importantly, the method can be fully validated and is overly sensitive and specific, identifying both known and unknown viruses and variants.
NGS can be used to detect viral genomes in fluids (virome analysis), endogenous viruses in cellular genomes (genome analysis), or virus-specific mRNA in cell substrates (transcriptome analysis).
Virome analysis is designed to detect all types of viral genomes across diverse virus families (ssDNA, dsDNA, ssRNA, dsRNA, segmented genomes, positive and negative sense genomes) in cell-free products. A transcriptome approach detects all kinds of viruses through virus-specific mRNA detection in cell-based products. Timely results can be delivered in two to three weeks.
For instance, cell lines such as CHO, Vero, HEK293, and Sf9 are standard materials for manufacturing biologics like recombinant proteins, viral vectors, and vaccines. Comprehensive screening for replication-competent viruses is crucial as low-level viral contamination can amplify significantly during cell culture. Transcriptome-based NGS offers a broad, agnostic method that detects known and novel variants, making it a more comprehensive viral safety tool than traditional approaches.
Like PCR, NGS can be used for many different approaches. “But the technology is a bit more complex because of the expertise needed to develop and interpret the bioinformatics pipeline. Our collaborator, PathoQuest, over time, has specifically developed bioinformatics for virus detection and identification in biologics,” Ruppach said. “The more experience you have, the more reliable and robust the analytics.”
Improving safety in cell therapies
New product modalities like cell therapy products, especially allogeneic products, have a higher risk than the recombinants, emphasized Ruppach. “In the future it will be mandatory to use NGS because it has the highest breadth of detection. All the other methods may fail in some certain cases. The risk is real even though there are so few products to date that we have not yet seen any contamination cases.”
Cell therapies, such as autologous and allogeneic CAR T therapies, present a unique viral safety challenge. Cells are administered directly to patients with no opportunity for the removal or inactivation of viral contaminants potentially introduced during manufacturing. Moreover, primary human cells or stem cells used to manufacture these therapies can carry endogenous viruses undetectable by traditional screening assays.
NGS detects any viral nucleic acid, independent of the virus’s ability to grow in indicator cells or animals. Transcriptome-based NGS identifies viral mRNA, confirming active or transcriptionally active latent viruses.
Master virus seeds used in producing recombinants, vaccines, vectors, oncolytic viruses, and virus-based products must be rigorously tested. Traditional in vivo and cell-based assays often struggle. Workarounds can reduce sensitivity, while neutralization with antibodies introduces contamination risks and is not always successful.
For these reasons, regulators allow testing of control cell cultures. This suboptimal solution does not test the product itself. The virome-targeting NGS approach is free from these constraints. It can test virus seeds or viral product batches directly, without dilution or neutralization, and still achieve high sensitivity despite the presence of product-specific nucleic acids, which can be identified and eliminated in the bioinformatics pipeline.
Comparing the new to the traditional
To demonstrate the benefits of using NGS, Charles River Laboratories and PathoQuest collaborated to perform side-by-side tests with several traditional viral detection methods using PathoQuest’s iDECT® Transcriptome assay.
Using mice and embryonated eggs, NGS exclusively detected four viruses. When compared directly against in vivo assays, it matched the sensitivity on another four and was less sensitive only for VSV in suckling mice, while still detecting one infected cell in a million non-infected cells.
A second replacement study tested the transcriptome-based NGS assay against MAP/HAP/RAP, and bovine/porcine virus 9CFR tests. In the MAP/HAP/RAP study, the transcriptome-based NGS assay detected all nineteen ICH Q5A (R2) rodent viruses and five additional ones, including both close and distant sequence variants. In the bovine/porcine virus 9CFR tests NGS identified thirteen additional bovine and porcine viruses beyond those required by regulatory guidance.
When compared to PCR, NGS detected all human and simian viruses analyzed in the study (thirteen viruses requested by regulatory guidance documents and fifteen additional human and simian viruses).
The 9CFR test, designed to detect specific bovine and porcine viruses is often supplemented with PCR to broaden detection. In contrast to PCR, NGS targets viral mRNA, allowing it to distinguish active infections and detect novel variants—broader coverage with comparable detection limits.
Regulatory momentumÂ
Regulatory momentum and economic advantages drive the push toward NGS for viral safety testing. Importantly, as noted earlier, the evolving regulatory landscape emphasizes the strengths of NGS and makes recommendations for its use.
Workflow improvements in automated sample prep, library prep, and analysis pipelines now allow for GMP-ready results with reduced hands-on time. The technology is well suited for AI implementation and will be further automated, speeding up the evaluation process. “Additionally, NGS does not require as much material as other assays,” added Ruppach.
NGS enhances a biologics manufacturer’s ability to detect and identify unknown or unexpected contaminants. This is specifically crucial for new product modalities where traditional testing does not address the factual viral risk.
Results are available in as little as two to three weeks and cell bank characterization programs can be reduced from three to four months to six weeks. “Five to six, and sometimes even seven, different methods are used to analyze a cell bank for viral safety to compensate the detection downsides of each assay,” explained Ruppach. “With NGS you can replace most of these, and in the end run two to three assays, one which is NGS, reducing costs and the timeline.”
Adopting an animal-free virus detection strategy aligns with global trends toward ethical testing under the 3Rs framework. Significant consolidated savings also result from replacing multiple redundant assays. In the long term, NGS will minimize repeat testing, compliance failures, and lower the operational burden of managing numerous testing platforms.
“I believe that due to the benefits, NGS will become a standard in virus testing in the future. In five years, people may wonder why we used the traditional approaches when we had this technology,” Ruppach concluded.
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