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University of Queensland Researchers Harness Nanopore Sequencing for mRNA Vaccine Quality Control


NEW YORK – Researchers from the Base mRNA facility of the University of Queensland in Australia and their collaborators have developed a nanopore sequencing-based workflow that promises to streamline quality control for mRNA vaccines and therapies.

Described in a study published in Nature Communications last month, the method, dubbed Vax-seq, analyzes key mRNA quality attributes, such as sequence identity, integrity, and contamination, by leveraging nanopore sequencing’s long-read and native RNA profiling capabilities.

The researchers recently announced a partnership with Oxford Nanopore Technologies to commercialize the technology for use by potential industry customers.

"For me, it's pretty intuitive that you would use RNA sequencing to analyze mRNA vaccines," said Tim Mercer, scientific director of the Base facility and the corresponding author of the study. "But that is not currently the industry standard."

According to Mercer, mRNA vaccine manufacturers currently deploy a mix of techniques — such as RT-qPCR, capillary and gel electrophoresis, liquid chromatography, and Sanger sequencing — to help determine the quality of their products. However, such methods can be "onerous and expensive to maintain, and often cannot sensitively detect key mRNA quality features," the study authors noted.

To overcome these limitations, the Base team turned to nanopore sequencing, which Mercer said has several advantages for analyzing mRNA molecules. For one, it allows researchers to sequence the entire length of the mRNA molecule, offering a way to determine the integrity of an mRNA vaccine, which is a key quality attribute.

"That is really key," Mercer pointed out. "You can imagine, as the mRNA vaccines are shipped around the world, they can get broken due to heat, RNases, or shearing forces. And if that is starting to impact the quality or the performance of the mRNA, you need to be able to detect that."

In addition, he said nanopore sequencing can detect off-target RNA contaminants with high sensitivity, which can be introduced as a result of in vitro transcription during the manufacturing process. It can also accurately validate poly(A) tails of mRNA molecules, which is key for ensuring stability and expression of the vaccines.

Beyond that, Mercer said, the real-time analysis capability of nanopore sequencing allows users to perform rapid quality-control testing within hours, a time frame that is desirable in an industrial setting. 

In the current study, the Base researchers deployed both full-length cDNA sequencing and direct RNA sequencing on the Oxford Nanopore platform. While the results showed that cDNA sequencing is currently "more reliable" than direct RNA sequencing, Mercer said the latter has the unique advantage of being able to directly measure RNA modifications. 

"When you are doing manufacturing, if you don't have to do that additional reverse transcription step, it does mean you are getting a bit of a truer measure of the pharmaceutical compounds," Mercer explained, adding that the ability to directly detect RNA modifications is crucial, since most mRNA vaccines now incorporate modified nucleotides.

To support Vax-seq, the researchers also developed a software tool called Mana that automatically analyzes mRNA quality.

Moreover, Mercer said the group has been validating the Vax-seq protocol to ensure it is "sufficiently robust" for industrial use. "When you are doing manufacture, it is a bit different to doing research; the methods you use need to be really carefully validated ​​to make sure that what you are seeing is really what you are getting," he noted.

This study "is formally showing what a lot of people in the R&D community thought should be feasible with direct RNA sequencing, and having a peer-reviewed proof in the literature is always a very encouraging thing," said Miten Jain, a bioengineering professor at Northeastern University who is an expert in nanopore sequencing but was not involved in this study. "I am mostly excited with the idea of being able to QC RNA vaccine candidates in companies or in academia by using direct RNA analysis."

While direct RNA nanopore sequencing can provide additional molecular insights compared with cDNA sequencing, Jain pointed out that one bottleneck for using the former has been the read accuracy. DNA nanopore sequencing is now achieving 99 percent or higher accuracy "consistently" in his lab, he said, while the accuracy of direct RNA sequencing still "has ways to go," lingering at around 90 percent in recent years. 

However, Jain said he sees "a genuine improvement" in the throughput and accuracy of Oxford Nanopore’s new direct RNA sequencing kit, RNA004, which was unveiled in May and which his lab has been using as an early-access customer. "We are seeing consistently 96 percent accuracy on RNA in our hands [with the new chemistry], which is quite remarkable, and the information on modifications is quite amazing," he noted.

Despite direct RNA sequencing’s promises, Jain said the field still needs to develop and train machine-learning models that can recognize and detect modifications of interest. Additionally, he thinks the ability to multiplex samples within one experiment could also be helpful for driving the technology’s adoption for mRNA vaccine analysis.

"The combination of the fact that you can do these in a multiplexed manner at good throughput and good quality, coupled with the cost, will start the adoption of those methods," he said.

Mirroring Jain’s point, Mercer said that RNA sequencing accuracy is important for Vax-seq, given that one of the key features of the workflow is to confirm the sequence identity. "Essentially, what that involves is improving the basecaller," he noted. "We have been working on that — it is still a bit of a work in progress."

To help advance and validate their method for commercial use, the Base team has partnered with Oxford Nanopore, which is providing in-kind contributions of sequencing consumables and technical support, as well as taking a lead in commercializing the workflow through the codevelopment deal.

"We are excited to work with the innovative Base team on methods for simplifying and ensuring the quality [control] of manufactured mRNA-based therapies using Oxford Nanopore’s direct RNA sequencing capabilities," Louisa Ludbrook, Oxford Nanopore's VP of commercial market development, wrote in an email. "Although we are still in the foothills of this research, we are pleased with how the work is progressing and see great potential for these methods to be applied more broadly in the industry, which aligns with the interest we have received from the market so far."

The company did not provide a commercialization timeline for the technology. 

In the long run, Mercer said the goal for the collaborators is to put together "a simple off-the-shelf, validated protocol" combining wet lab and analysis workflows that can be readily deployed for the mRNA vaccine industry.

"In manufacture, the analysis has to be very simple, very reproducible, very accurate, and robust," Mercer said. "In an ideal world, you could run your analysis with a simple command, which can essentially put out an automated report of what the mRNA vaccine quality is."