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Study Shows Promise of NGS, qPCR to Detect Vaccine Contaminants

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NEW YORK (GenomeWeb) – Next-generation sequencing and real-time PCR could detect adventitious agents in vaccines including human cytomegalovirus more quickly and at lower thresholds than existing cell-based methods, according to a recently published proof-of-concept study.

Scientists from Sanofi Pasteur, led by Siemon Ng, ran parallel studies comparing NGS and qPCR detection tests to the established in vitro cell culture method for CMV detection.

"From an industrial perspective, the conventional in vitro and in vivo assays used for detection of viral contaminants have shown their limitations," Sanofi Pasteur's Laurent Mallet and Lucy Gisonni-Lex, co-authors on the paper, wrote in a separate article in the same journal issue. 

Researchers have seriously considered using NGS to detect contaminants since 2010, when scientists discovered a nonpathogenic pig virus in Rotarix, the GlaxoSmithKline rotavirus vaccine.  

"Our results show that NGS can potentially achieve a detection limit that is 10 times more sensitive than the cell culture assays," the authors said. The cell culture assay detection limit was 10 CCID50/mL.

NGS was able to detect CMV at a sensitivity of 1 CCID50/mL and was able to detect the virus directly at initial levels of inoculation of 10 and 100 CCID50/mL, which could eliminate the need for cell culture to amplify the virus, reducing the time it takes to detect CMV.

The qPCR test detected CMV at a sensitivity between 1 and 10 CCID50/mL. "It still remains to be worked out where qPCR can go. If you optimize qPCR you might be able to get [the detection threshold] down," Ng said. The authors said that qPCR could also detect CMV at levels that would obviate the viral amplification step.

The results were originally presented in November 2013 at the Parenteral Drug Association/US Food and Drug Administration Advanced Technologies for Virus Detection in the Evaluation of Biologicals Conference and were published in the November/December 2014 issue of PDA Journal of Pharmaceutical Science and Technology.

CMV, related to herpes viruses, is highly prevalent in the general population and can pose a serious health risk to newborns and other individuals with compromised immune systems. It represents a class of potential contaminants in cell lines used to manufacture biological products like vaccines.

The FDA Guidance for Industry specifically calls for human diploid cells to be used in the detection of adventitious agents such as CMV, which requires inoculating cell lines and incubating the cultures for 28 days, after which a technician checks for signs of viral infection.

For the NGS study, the scientists used the Illumina Nextera XT DNA sample preparation kit to generate the libraries for each extracted sample and performed sequencing with the HiSeq 1500 system. The authors noted that lower throughput NGS systems might miss the viral signal.

For the parallel qPCR study, the scientists extracted nucleic acid with the Wako DNA extraction kit. The qPCR reactions took place on the Roche LightCycler 480 system.

Detecting CMV or other adventitious viruses with NGS is not without its limitations. NGS is likely to take longer than a qPCR assay and detection of viral sequences does not necessarily guarantee that the virus present in the sample is able to replicate.

But NGS offers the ability to detect other potential adventitious viruses, without having to look for them specifically. "In the same sequencing run I could detect CMV or whatever other potential contaminant is in there," Ng said. "[NGS and qPCR] can be consider complementary as NGS (higher in cost and time) might be run at specific stage of production while qPCR can be more of a routine testing protocol."

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