An Updated Approach to mRNA Vaccine Quality Assessment

A vial labeled “mRNA vaccine” and a syringe on a blue background.

Mercer’s group recognized benefits of cDNA-based long-read nanopore sequencing for measuring mRNA vaccine integrity.

A well-earned highlight shines on mRNA vaccines, with current Nobel prize accolades awarded to Katalin Karikó and Drew Weissman for his or her foundational analysis that enabled speedy and efficient vaccine growth throughout the covid pandemic. As mRNA vaccine analysis picks up velocity in purposes past covid immunization, instruments that permit scientists to enhance their manufacturing strategies are more and more essential. Till not too long ago, vaccine producers largely relied on strategies equivalent to gel electrophoresis and liquid chromatography, which researchers first adopted for protein-based vaccines and prescription drugs that held middle stage previous to the pandemic.1 Though strong and dependable, these high quality management strategies are time-consuming and expensive for analyzing mRNA vaccine merchandise. 

“Throughout the pandemic, clearly due to the accelerated velocity, plenty of these processes had been simply inherited from conventional producer workflows,” defined Timothy Mercer, an RNA biologist on the College of Queensland who leads an mRNA manufacturing facility constructed on artificial biology, genomics, and transcriptomics analysis. “The precise product itself obtained moved so much sooner than the ecosystem round it, which is now within the means of catching up.” 

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The precise product itself obtained moved so much sooner than the ecosystem round it, which is now within the means of catching up.
-Timothy Mercer, College of Queensland

For Mercer, bringing sequencing strategies into that ecosystem was an intuitive resolution for bettering high quality management analyses. Scientists exterior the vaccine manufacturing area steadily use sequencing to investigate features of DNA and RNA biology, together with gene expression, RNA polyadenylation or poly(A) tails, and post-transcriptional chemical modifications that have an effect on RNA stability and immune activation.2 

In Mercer’s newest work revealed in Nature Communications, his group developed a streamlined methodology referred to as VAX-seq to investigate mRNA vaccines utilizing long-read nanopore sequencing.3 Their methodology measures key vaccine high quality attributes at completely different manufacturing steps, together with sequence id, integrity, and contamination.

The researchers investigated and in contrast a number of sequencing approaches, together with short- and long-read sequencing of cDNA that they generated from the mRNA vaccine and direct RNA sequencing with long-read nanopore expertise. Mercer’s group successfully analyzed vaccine sequences with every approach and recognized cDNA-based long-read sequencing as the perfect method for measuring vaccine high quality. In contrast to short-read strategies, long-read nanopore sequencing was efficient for full-length and fragmented mRNAs, which offered perception into mRNA degradation. “Since you’re sequencing the entire molecule, you are not simply figuring out what the sequence of that molecule is, however you are additionally saying it is a full-length practical molecule,” Mercer mentioned. “That is an extra high quality metric, which is admittedly key to the precise pharmaceutical exercise of that specific mRNA pattern.” 

This benefit aligns with long-read sequencing’s recognized strengths. “I feel that the strategies they’ve give you are very present and of curiosity.” mentioned Cynthia Burrows, a chemist from the College of Utah who’s a nucleic acids professional and who was not concerned within the examine. “As a scientist, what you’ll wish to know is, are most of your strands full size or has there been some aborted synthesis? Does all of it have the poly(A) tail on the finish? These are the kinds of issues that the paper addressed.”

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Though Mercer’s group discovered cDNA sequencing extra dependable than direct RNA sequencing, they count on that long-read RNA sequencing will proceed to be a helpful analysis instrument for mRNA vaccine growth. For example, Burrows and different researchers have already used direct RNA sequencing to analyze the properties of nucleoside modifications,4 such because the pseudouridine modification in mRNA vaccines that garnered Katalin Karikó’s and Drew Weissman’s Nobel recognition. “That was to this point forward of its time, and the battles that had been fought there to maintain shifting ahead and hold the science going in order that swiftly, once we actually wanted mRNA vaccines, they had been there for us. That may be a phenomenal story,” Burrows mentioned. 

Sequencing mRNA vaccines is one other vital chapter in that story, shifting the science ahead by connecting technical developments with translational breakthroughs. Helen Gunter, the postdoctoral fellow in Mercer’s laboratory who spearheaded this work, is already increasing VAX-seq previous high quality management purposes by collaborating with different analysis labs all through Australia. “She’s beginning to get a extremely good understanding throughout all these completely different mannequin techniques, all these completely different cells, concerning the range of the way during which the enter, the RNA, is definitely taken up into the cell, processed, and expressed,” mentioned Mercer. “I feel coming from that organic perspective, the sequencing is a extremely great tool.”


  1. Rosa SS, et al. mRNA vaccines manufacturing: Challenges and bottlenecks. Vaccine. 2021;39(16):2190-200.
  2. Baronti L, et al. A guide to large-scale RNA sample preparation. Anal Bioanal Chem. 2018;410(14):3239-52.
  3. Gunter HM, et al. mRNA vaccine quality analysis using RNA sequencing. Nat Commun. 2023;14(1):5663.
  4. Fleming AM, Burrows CJ. Nanopore sequencing for N1-methylpseudouridine in RNA reveals sequence-dependent discrimination of the modified nucleotide triphosphate during transcription. Nucleic Acids Res. 2023;51(4):1914-26.

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