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Biologics
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Audrey Brussel, PhD, PathoQuest

The Emergence of Next Gen Sequencing for Analyzing Biologics

DNA sequencing began in the 1980s as an exquisitely tedious process used primarily to sequence individual genes and small viral genomes. But the technology quickly evolved into something bigger, and BIGGER and BIGGER STILL—ever faster, cheaper and more accurate methods of deciphering both DNA and RNA sequences. Today, it is an essential tool in scientific data generation but retains the name “next-generation” because it is continually showing off new tricks.

In the realm of biologics testing—the subject of this blog— next gen sequencing (NGS) has emerged as an analytical tool to analyze vaccines, monoclonal antibodies and other products for the presence of unwanted viral contaminants. Biologics can be as finicky as Mother Nature herself. One of the biggest challenges are identifying the presence of adventitious viruses --industry-speak for viruses that unintentionally get introduced into the manufacturing process.

NGS: Myth vs. Fact

Historically, laboratories have turned to a combination of in vivo , in vitro and PCR—a tool that amplifies a segment of DNA—to track these unwanted microorganisms. But next-gen sequencing methods are being used more and more in virus testing, both as an adjunct and replacement for traditional method that, in some cases, falls short.

The contamination of a rotavirus vaccine over a decade ago demonstrates why we need alternative avenues for tracking adventitious agents. The pediatric vaccine, given to prevent a common cause of diarrheal disease in infants, was on the market in the US and EU when an NGS tool uncovered a pig virus that had been in the vaccine since it was developed! While the virus was not considered a public health threat, it was still concerning that it had gone unnoticed for years.

NGS has been subjected to a number of myths –that it detects too many signals, that we do not know its sensitivity or breadth of detection, that it is not a GMP assay or validated—but the reality is something quite different.

NGS doesn’t just provide positive signals. Innovative NGS approaches have been developed that can detect live viral contaminants in cells. Its performance has been compared favorably to traditional assays, such as PCR or in vivo assays, demonstrating similar or high sensitivity. NGS is also amenable to validation and GMP compliance, which is why regulatory bodies are reviewing and updating regulatory guidance and requirements to allow the use of NGS for virus detection. The chart below compares nicely the differences between NGS and traditional assays.

Chart comparing PCR/In Vivo with NextGen method

Both the US Food and Drug Administration, the World Health Organization and the European Pharmacopoeia already provide flexibility for using “fit-for-purpose” alternative approaches. Last year, the value of NGS as one of these alternative approaches was realized when there was a tremendous need to accelerate the development of SARS-CoV-2 vaccines. Not surprisingly, the FDA received multiple requests from sponsors interested in using NGS.

Can NGS identify replicating viruses?

One of the challenges of NGS has been its inability to differentiate sequences from replicating viruses vs. background inert sequences. But a recent collaboration that included our lab and Charles River Laboratories, found a workaround to this. We designed a new NGS approach that targets subsets of viral RNAs only synthesized during cell infection. Then we tested this approach in two different virus/cell systems. The findings, published in 2019 in Biologicalsfound the sensitivity of our RNA NGS approach to be equivalent to traditional PCR with an increased specificity for live, infectious viruses. Our system also identified a previously undetected murine leukemia virus contaminant in the cell system we used, well illustrating the increased breadth of detection of NGS.

Of course, there are still challenges, notably regarding regulatory acceptance worldwide. The current International Council for Harmonisation (ICH) guideline Q5A is currently being updated to include NGS-based tests to assess viral safety and its release is expected in November 2022. The adoption of such tests will remain an incremental process, and the pace may vary between countries. As manufacturers will be faced with this discrepancy in NGS-based test adoption, it may delay the broad use of the NGS-based approach as a prime method for viral safety testing for biologics. Still, it is reliable and durable enough to replace in vivo tests in the long run, to improve biosafety and accelerate drug disposition.

Looking ahead, one could imagine the application of NGS in portable closed devices, plugged into bioreactors, continuously sequencing RNA directly in real-time, cloud computing of the sequence data, ultimately enabling the real-time release of drug products.

And the evolution of sequencing marches on, as it has for 50 years.

Audrey Brussel, Ph.D, is a Virus Safety Leader with PathoQuest, which provides NGS-based testing solutions for clients. PathoQuest has been a strategic partner of Charles River Laboratories since 2016.