Literature Review

mRNA vaccines are a new vaccine modality which utilise synthetic messenger RNA (mRNA) which has been engineered to mimic a mature eukaryotic mRNA transcript encoding a specific protein which is capable of causing a therapeutic effect. These medicines are being developed for the treatment of various diseases including cancers, infectious diseases, genetic diseases, and autoimmune diseases (Parhiz et al., 2024).

In the case of the various COVID-19 mRNA vaccines, this specific protein which is being produced is the SARS-CoV-2 spike protein, a viral antigen which causes an immune response in the patient. mRNA vaccines are important medical treatments because they are faster to design and manufacture than conventional vaccines making them excellent for the fight against rapidly emerging diseases, as was seen in the response to the COVID-19 pandemic in 2020 (Slaoui and Hepburn, 2020).

This review examines the literature on assessing mRNA stability alongside other quality characteristics using the Next-Generation Sequencing (NGS) technique of Oxford Nanopore Sequencing (ONS). The mechanisms of mRNA degradation and the importance of mRNA stability for vaccine efficacy and safety will also be discussed. ONS is evaluated alongside both traditional non-sequencing methods and other NGS approaches used for analysing mRNA stability. This review outlines the advantages and limitations associated with ONS compared to these methods and addresses the current challenges and future directions for integrating ONS into mRNA vaccine quality analysis workflows.

2.1. Importance of Stability in mRNA Vaccines 

mRNA stability refers to the lifespan of an mRNA molecule before it is degraded. Stable mRNA is crucial for effective protein expression, and rapid degradation of the molecule leads to reduced translation output affecting vaccine efficacy. 

The rapid success of mRNA vaccines during the COVID-19 pandemic has highlighted the need for robust analytical tools to ensure vaccine quality and stability. Measuring the stability of mRNA vaccines is key to ensuring that the product maintains its efficacy in vivo. Cleavage of the mRNA strand or base modifications which may occur during manufacture can have unfavourable effects on the quality profile of the product and so strict quality control must be performed throughout the various stages in mRNA vaccine manufacturing, from initial plasmid preparation to final product characterisation (Gunter et al., 2023).

mRNA vaccines typically include various structural elements which improve the stability of the polynucleotide. These include a 5’ cap, a 5’ untranslated region (UTR), a 3’ UTR, and a 3’ poly(A) tail (Figure 7). These structural elements are important as they help maintain the mRNA transcript’s stability and also prevent degradation. Despite these inclusions, mRNA strands remain highly fragile molecules which are extremely susceptible to enzymatic degradation.

mRNA molecules are inherently sensitive, they are large, negatively charged, and prone to degradation, so maintaining their stability (both chemical and physical) is crucial for an effective vaccine. The UTR regions may have stabilising elements included within their sequence or even protect the vulnerable regions of the mRNA molecules through their secondary structure conformation (Stephenson et al., 2022). Close monitoring of these specific structural elements is integral to measuring mRNA vaccine stability. 

The greatest threat to mRNA vaccine stability is the presence of RNases, ubiquitous nuclease enzymes which catalyse the degradation of RNA. These enzymes are present everywhere and can rapidly cleave mRNA, leading to reduced integrity of the mRNA strand. For this reason, mRNA vaccines must be manufactured in completely RNase-free conditions, this necessitates strict control over all reagents, equipment, and environments to ensure the absence of RNases. 

2.2. Manufacturing and Formulation of mRNA Vaccines 

mRNA vaccines are produced through in vitro transcription (IVT). This process involves transcription of mRNA from DNA plasmid templates using RNA polymerase. The purity of mRNA produced in this way is crucial and so the purification process will usually involve several chromatographic techniques in order to remove impurities or incomplete transcripts (Schlake et al., 2012) (Figure 8). The formulation of mRNA vaccines commonly involves encapsulation of the mRNA strand into lipid nanoparticles (LNPs) in order to protect the mRNA molecules from RNase-mediated degradation and also in order to allow for easier cellular uptake. LNP formulation significantly impacts the stability, efficacy, and safety of the vaccine product (Schoenmaker et al., 2021).

LNPs protect encapsulated mRNAs from degradation by RNases but the components of LNPs may also catalyse mRNA degradation over time. For example. In a computational study of mRNA stability, Wayment-Steele et al. predicted a 100-fold increase in mRNA strand autocleavage when it is encapsulated with cationic lipids (Wayment-Steele et al., 2021). Cationic lipids are used in LNP mRNA formulations in order to facilitate the delivery of negatively-charged mRNA strands to host cells (Crommelin et al., 2021).

Quality control procedures measuring mRNA stability, identity and other important variables must be implemented throughout the manufacturing process as well as on the final drug product in order to ensure acceptable vaccine efficacy and safety in vivo.

2.3. Quality Control of mRNA Vaccines

The stability and integrity of mRNA sequences are critical quality attributes (CQAs) in the development and manufacture of mRNA vaccines. However, several other CQAs unique to mRNA vaccines must be monitored in order to fully assess their quality profile including poly(A) tail quality and capping efficiency (Camperi et al., 2025).

Vaccines require stringent quality control measures to ensure their efficacy and safety in vivo. One of the primary concerns in mRNA manufacturing is the presence of contaminating products such as truncated transcripts, double-stranded RNA (dsRNA), and other impurities that may arise during IVT. These impurities may cause adverse immune responses, or they may decrease the efficacy of the treatment and so robust analytical tools must be used to quantify the extent of these contaminants (Huang et al., 2024).

In particular, stability of mRNA vaccines is of great interest as a CQA. If the mRNA molecule has its stability jeopardised either by RNase digestion or strand autocleavage, it may fail to produce the intended polypeptide, compromising the desired therapeutic effect. mRNA stability may be measured a number of different ways. The key quality attributes of mRNA vaccines, sequence identity, length integrity, poly(A) tail length, and purity may all be assessed using ONS (Gunter et al., 2023).

The mRNA Poly(A) tail is a long sequence of successive adenine residues which are present on the 3’ end of the mRNA molecule. Poly(A) tail length is a quality characteristic of particular interest as it plays an integral role in the stability and translation efficiency of mRNA vaccines (Brouze et al., 2023). 

2.4. Traditional Methods of mRNA Quality Analysis

Traditionally, mRNA quality analysis is carried out using a diverse range of analytical assays. Techniques such as Reverse Transcriptase PCR (RT-PCR), Sanger sequencing, gel electrophoresis and HPLC have been used in order to characterise various quality characteristics of mRNA vaccines however, these techniques are often time-consuming, costly and labour intensive (USP-NF, 2022).

For measuring the integrity and purity of mRNA transcripts, tools such as Agilent’s Fragment Bioanalyser and capillary gel electrophoresis (CGE) are commonly used. These tools evaluate RNA lengths and size distribution (Camperi et al., 2025). CGE is a high-resolution method commonly used to evaluate truncated mRNA transcripts and other degradation products. CGE separates full-length mRNA transcripts from impurities based on their distinct hydrodynamic radii and their differential migration through a gel solution. The different species are detected following separation using either laser-induced fluorescence (LIF) or UV absorption assays. 

While widely used for mRNA size evaluation, CGE is limited by its relatively long separation times, usually taking between 40 and 130 minutes (Camperi et al., 2025). Clearly, a faster analytical technique is required for mRNA length analysis if mRNA vaccine manufacturing is to become more commonplace. 

2.5. Oxford Nanopore Sequencing Technology 

ONS is a novel sequencing technology which utilises a unique method of analysing nucleic acids. ONS detects single-stranded RNA/DNA molecules as they pass through a nanoscale pore by monitoring the ionic current in the pore. In a typical nanopore sequencer, there is an array of pores embedded in a small flow cell through which the single stranded polynucleotide is pulled through once an electric potential is applied across the membrane. Each distinct nucleotide produces a characteristic signal which is detected by the device (basecaller) and decoded in order to sequence the polynucleotide strand. This presents a promising alternative for stability analysis as it is an accurate and high-throughput sequencing technology (Wang et al., 2021).

ONS is considered a Next-Generation Sequencing (NGS) technology alongside other methods such as Illumina and PacBio. These NGS methods are capable of revealing insights into various CQAs such as sequence identity, purity, nucleoside modifications, and purity. Each of these advanced NGS techniques display unique advantages, for example, Illumina is excellent for short read sequencing and accuracy, while ONS conserves native mRNA modifications by using direct RNA sequencing (Camperi et al., 2025). 

ONS produces long reads of RNA/DNA strands as opposed to the short reads produced by other NGS methods such as Illumina sequencing. Another advantage to using ONS for RNA sequencing is the fact that it does not require the RNA to be converted to DNA and that it may be sequenced natively, this is known as direct RNA sequencing (Gunter et al., 2023). 

2.6. Use of ONS for Analysis of mRNA Vaccine Stability

The use of ONS in mRNA vaccine stability analysis has clear logistical advantages over the traditional analytical methods but it is important to compare these methods in terms of sensitivity and accuracy in order to make a fair judgement. Traditional qPCR can be extremely sensitive for specific targets, such as known DNA contaminants, while ONS has the unique advantage of being able to detect even unanticipated issues simply because of the nature of the technique i.e., its ability to sequence any DNA/RNA present in the sample (Camperi et al., 2025). 

ONS has an advantage over the other aforementioned NGS technologies because it avoids the errors which may accumulate during the PCR amplification steps inherent in those methods. Direct RNA sequencing does not require the synthesis of a cDNA (complementary DNA) strand in order to sequence mRNA and so it is theoretically capable of greater read fidelity (Wang et al., 2021), even though there is a protocol for direct ONS sequencing of cDNA generated from mRNA templates. However, a study from Chen et al. (2021) comparing Oxford Nanopore directly sequenced RNA, Oxford Nanopore directly sequenced cDNA, and PCR-amplified cDNA for the identification of gene isoforms found that they all perform similarly.

Traditional methods do have an advantage in their reliability, Gunter et al. found that cDNA (complementary DNA) sequencing was more reliable than their newly-developed, ONS-based VAX seq workflow (Gunter et al., 2023). However, several inherent features of nanopore sequencing make it particularly well suited for analysing mRNA vaccine stability and quality. Using full-length sequencing, a wealth of important information about the molecule of interest can be gathered. For example, by sequencing the entire RNA molecule the assay discovers not only the sequence/identity of the molecule, but it also determines that it is a full length molecule which is another important quality characteristic.

One of the most notable studies in this area of research is without a doubt that carried out by Gunter et al. (2023). This study presents a comprehensive mRNA vaccine quality analysis workflow which uses ONS to measure various key vaccine quality attributes namely, sequence, length, integrity, and purity. In this study, the researchers compared their novel VAX-seq workflow to the industry standard techniques of agarose gel and capillary electrophoresis methods using a model mRNA sequence (eGFP). Of particular note, they found that mRNA size distribution (77% full length mRNAs and 23% fragmented mRNAs) measured using the VAX-seq protocol was analogous to the mRNA size distribution measured using capillary electrophoretic methods (Gunter et al., 2023).

Gunter et al. (2023) provides the most comprehensive assessment of ONS as a tool for mRNA vaccine quality analysis. Crucially, the researchers used VAX-seq to measure the integrity profile of the mRNA, by plotting read length distribution. This distribution allows manufacturers to track how much of the sample was full-length or truncated. This quality analysis workflow may be employed at various points throughout the mRNA vaccine manufacturing process and allow manufacturers to accurately assess mRNA stability during manufacture, storage, or delivery.

2.7. Limitations of ONS for mRNA Stability Analysis

One of the main problems with using ONS-based direct mRNA sequencing for the quality analysis of mRNA vaccines is the fact that ONS does not perform well when reporting the lengths of poly(A) tails. This is because the basecaller cannot distinguish between the signals from individual bases in long stretches of “homopolymers” such as the poly(A) tail in mRNA. Computational techniques such as the R package tailfindr have been developed in order to estimate the lengths of poly(A) tails from ONS-based data (Krause et al., 2019), however, this still presents a significant limitation of the ONS platform. 

Direct RNA sequencing is also capable of characterising off-target RNA contaminants generated during in vitro transcription (Gunter et al., 2023), an important quality metric which must be removed in order to ensure vaccine safety and efficacy (Baiersdörfer et al., 2019). These off-target RNAs will not display the desired therapeutic effect in the patient and so their characterisation and quantification must be monitored closely as they pose a serious risk to the quality of the product.

Nanopore sequencing is also capable of detecting modified nucleosides such as N1 methylpseudouridine which is extremely important for vaccine applications, as these bases are incorporated in order to lessen the immune response towards exogenous mRNA (Karikó et al., 2005). Direct RNA sequencing allows for confirmation of the presence of these modified bases which is extremely relevant for the quality analysis of mRNA vaccines using these bases. Traditional methods such as mass spectrometry are capable of measuring overall modification content but are unable to pinpoint exactly where these modifications occur (Camperi et al., 2025).

Beyond the primary structure of mRNA, the secondary structure of the molecule may also influence its stability. Nanopore sequencing has been recently combined with chemical probing by Stephenson and colleagues in order to create a technique known as nanoSHAPE which may allow researchers to infer the secondary structure of the molecule (Stephenson et al., 2022). This method allows researchers to further characterise the stability profile of the mRNA strand, providing a more complete picture of the factors influencing mRNA stability.

Within the area of study on NGS technologies being used to assess mRNA stability, there are some contrasting approaches which may be compared. 

ONS vs Short-read sequencing 

Short-read sequencing of mRNA using Illumina technology has long been considered the gold standard for high fidelity sequencing of mRNA transcripts when used in transcriptomic research. However, it cannot assess full-length mRNA structure like direct RNA sequencing (ONS). For example, if a fragmented mRNA transcript is read using Illumina, it is not possible to discern whether the 3’ end or 5’ end was lost. On the other hand, if a fragmented mRNA strand is pulled through a nanopore, it may be easily determined at what point in the strand the cleavage occurred (Chen et al., 2021). 

ONS cDNA vs ONS Direct RNA

ONS is capable of sequencing native RNA directly or sequencing cDNA which has been synthesised using reverse transcriptase. cDNA sequencing may be required if the sample of mRNA available is a low amount, but it loses details of the native mRNA features such as the presence of modified nucleosides. 

The stability of mRNA is a critical quality attribute which determines the efficacy, safety, and quality of mRNA vaccines. As the market for mRNA continues to expand, there is a growing need for the implementation of a robust, accurate, and high-throughput analytical tool which can fully characterise the stability of mRNA molecules. Oxford Nanopore Sequencing provides a comprehensive and versatile platform which addresses many of the limitations associated with traditional and short-read sequencing technologies. Its ability to directly sequence full-length mRNA molecules, preserve native mRNA modifications, and detect mRNA sequence length makes it a great candidate capable of assessing key CQAs such as poly(A) tail length, off target transcripts, and modified nucleotides.

The development of various bioinformatic techniques such as tailfindr and nanoSHAPE among others, which have been developed alongside ONS, may allow us to accurately and completely characterise mRNA vaccine stability. The implementation of high-throughput Next Generation Sequencing techniques into the mRNA vaccine manufacturing workflow seems to be the logical next step as mRNA vaccines are becoming increasingly popular as next-generation therapeutics.