mRNA vaccines have redefined the future of medicine, turning our cells into programmable factories for fast, precise, and adaptable immune defense. Their ability to be rapidly designed and scaled has enabled unprecedented speed in responding to global health threats, such as COVID-19. But this promise comes with a challenge: ensuring safety and efficacy demands accurate measurement of critical mRNA features. Yet, traditional quality control remains fragmented, slow, and labor-intensive, threatening to bottleneck innovation at the very point it's needed most.
Long-read sequencing technologies are offering a streamlined, end-to-end solution for assessing critical quality attributes essential to mRNA vaccine safety and efficacy. In this article, we explore how long-read Nanopore sequencing is being applied in the field to enhance mRNA vaccine quality control (QC), delivering comprehensive RNA analysis in a single assay, accelerating development, ensuring consistency, and supporting regulatory readiness at scale.
The effectiveness of mRNA vaccines depends on key transcript features such as the 5′ cap, poly(A) tail, nucleotide modifications, and overall mRNA purity, which are essential for efficient translation and ultimately, vaccine function. Deficiencies in these 'self'-identifying elements or the presence of contaminants like double-stranded RNA or truncated transcripts can reduce vaccine efficacy and, in severe cases, provoke harmful immune responses with lasting adverse effects 1–4 . To safeguard quality, these attributes are rigorously evaluated during quality control. However, standard QC workflows typically rely on multiple separate assays to assess individual features, making the process slow, labor-intensive, and often limited in accuracy, reproducibility, and throughput.
The protective 5′-cap is vital for mRNA vaccine effectiveness and stability. Without it, transcripts are unable to recruit the host machinery to initiate and sustain translation and are susceptible to exonuclease degradation5. Similarly, the length of the poly(A) tail is a critical determinant of mRNA stability and translational efficiency, as transcripts with absent or short tails (15–30 nucleotides) show significantly reduced protein production6.
Epitranscriptomic modifications, such pseudouridine (Ψ), is also extremely important to reduce transcript immunogenicity while enhancing stability and translational capacity. Modified mRNA has been shown to dramatically increase therapeutic protein production in animal models, while the unmodified version, despite having the same sequence, failed to produce meaningful expression and instead triggered a strong immune response 7.
Finally, process-related impurities that arise during the in vitro transcription process, such as aborted truncated transcripts and double-stranded RNA, can blunt vaccine efficacy and provoke unintended immune activation, driving side effects like fever, myalgia, and other reactogenic symptoms.
To assess each of these critical attributes, multiple assays need to be utilized. Electrophoresis and qRT-PCR effectively measure mRNA capping efficiency while liquid chromatography–mass spectrometry (LC-MS/MS) is considered the gold standard for modified nucleotide content assessment and poly A tail length determination8–11. Assays like ELISA and capillary gel electrophoresis are used to detect process-related impurities12.
Together, these workflows are slow, costly, and variable, and often miss critical transcript features, which compromises vaccine quality and performance. LC-MS/MS, although considered the gold standard for nucleoside detection, requires RNA to be fully digested into individual nucleosides, destroying sequence context and positional information of these critical modified nucleotides 9–11,13. Similarly, ELISA and capillary electrophoresis, both multistep assays with notable assay-to-assay variability, can show inconsistent sensitivity in detecting double-stranded RNA (dsRNA), especially when dsRNA species vary in size. This variability can lead to incomplete detection of harmful contaminants, posing potential risks to vaccine safety 12.
Key mRNA features like the 5′-cap, poly(A) tail, and nucleotide modifications are vital for vaccine effectiveness. However, current testing methods are limited by complexity and sensitivity, risking compromised quality and safety due to undetected impurities and undefined biology. Improving these assays is crucial to ensure safe, effective mRNA vaccines
Long-read sequencing offers a high-resolution, end-to-end view of mRNA quality, enabling faster, more efficient analysis across development and manufacturing. Its all-in-one approach improves lot-to-lot consistency and reveals critical biological insights, driving innovation and accelerating the creation of safer, more effective mRNA therapeutics.
One recent study combined reverse-transcribed cDNA reads to provide accurate assessment of sequence fidelity, transcript abundance, and template-derived impurities. Direct RNA sequencing complemented this with single-molecule resolution of poly(A) tail length, nucleotide modifications, capping efficiency, and detection of contaminants such as double-stranded RNA and residual DNA, delivering analytical depth on par with industry standards like chromatography and electrophoresis 14.
Another breakthrough study confirmed that nanopore sequencing not only supports comprehensive mRNA QC data for routine manufacturing but also empowers ongoing mRNA manufacturing process optimization to enhance yield and product quality, positioning it as an indispensable tool for current and next-generation mRNA vaccines and therapeutics15.
Long-read sequencing enables comprehensive, single-assay analysis of critical mRNA quality attributes, including capping efficiency, sequence fidelity, poly(A) tail length, nucleotide modifications, and purity, with sensitivity comparable to industry-standard methods. Oxford Nanopore’s real-time, scalable platform supports both process development and lot release, accelerating vaccine production and high-quality therapeutic development. With 5′ cap detection technology currently in development, ONT is advancing toward a fully integrated, end-to-end solution for mRNA QC positioned to streamline workflows and strengthen regulatory confidence in long-read sequencing for vaccine manufacturing.
In an era where mRNA vaccines must be developed, produced, and released with unprecedented speed and precision, robust and comprehensive quality control is essential. Traditional analytical methods, while sufficient in determining these critical quality attributes, are often fragmented, labor-intensive, and limited in sensitivity, posing risks to vaccine safety, efficacy, and regulatory approval. Long-read nanopore sequencing addresses these challenges by enabling single-assay, high-resolution analysis of all critical mRNA quality attributes, from 5′ capping and poly(A) tail length to nucleotide modifications and process-related impurities. Its scalability, real-time readout, and compatibility with both early-stage development and GMP-compliant lot release position it as a transformative solution in mRNA vaccine manufacturing, accelerating safer delivery of high-quality therapeutics to patients worldwide.
