By Vivienne Raper, PhD

Scientists have designed a new method for detecting and analyzing double-stranded RNA during the manufacture of messenger RNA products. The team of Anubhav Tripathi, PhD, from Brown University, claim the technique is cheap and uses equipment commonly available in laboratories and industry.

The aim is to address concerns over tiny amounts of double-stranded RNA (dsRNA) impurities that form during the manufacture of vaccines and other drugs. These can generate a harmful immune response in patients. According to Adriana Coll De Peña, a PhD candidate in biomedical engineering at Brown, “The reason we care about dsRNA is that it can be found in viruses, but it’s not produced by our own bodies, so they see it as a threat.”

Graphical representation of the dsRNA contaminant analytical method developed by the Tripathi Lab.      [Nina Li]
For this reason, the WHO and other organizations have highlighted the importance of purifying mRNA therapies of dsRNA during manufacturing. Unfortunately, according to De Peña, existing techniques such as ELISA assays can only reveal the total amount of dsRNA present and not, for example, the lengths.

Other analytical techniques, such as gel electrophoresis, are unsuitable for dsRNA because of the low concentrations, she says, noting that the length of dsRNA, has been linked in some publications with the severity of the undesirable immune response. To overcome this problem, De Peña and colleagues decided to develop their own microfluidic assay to detect the amount, and length, of dsRNA present in an mRNA sample.

Two different fluorescent dyes

After many experiments, the team settled on using two different fluorescent dyes—one for labeling RNA and another for DNA. Because of the double-stranded structure of dsRNA, the team reasoned that it would respond to the dye like double-helix DNA.

The researchers found that, by running the samples independently with each dye, they could detect which RNA was dsRNA by whether it responded more to the DNA or RNA dye. The mRNA in the sample responded more strongly to the RNA label, De Peña explained, whereas the dsRNA had a higher affinity to the DNA dye.

Once they detected the dsRNA, they were able to use a dsRNA ladder, a sample with varying, known dsRNA fragments, to estimate the lengths of the dsRNA contaminants.

De Peña explained the technique can be incorporated into existing manufacturing processes without needing specialist equipment.

“The building blocks often already exist at a company,” she tells GEN. “So, this is a relatively cheap, easily available method that a company can take away and do right now provided that they understand how to implement it into their processes.

“The key advantage of this method is that it’s fast, high-throughput and, in addition to identifying [dsRNA), you can use it to determine size.”

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