Improving the analysis of RNA medicines: what to expect in 2024

Published: 26-Jan-2024

In vitro transcribed (IVT) RNA development has advanced since the first messenger RNA (mRNA) vaccines to prevent COVID-19 entered the market in 2020. Now the focus of similar drug candidates is on other prophylactic vaccines to prevent infectious diseases, including influenza and respiratory syncytial virus (RSV), and therapeutic drugs that might soon be used to treat cancer and other diseases

These RNA developments are enabled by technologies that allow researchers to determine the purity, stability and other key characteristics that are essential to both safety and efficacy.

Investments into IVT RNA research and development during the past few years have enabled drug candidates to expand rapidly; beyond regular mRNA, two other modalities are also gaining steam: circular RNA (circRNA) and self-amplifying RNA (saRNA) or self-replicating RNA (srRNA). 

Kerstin Pohl, Senior Global Manager of Cell and Gene Therapy and Nucleic Acids at SCIEX, discusses both the opportunities and challenges of emerging RNA modalities.

The analytical hurdles vary across the three modalities, making the development of new drugs particularly challenging. However, the good news is that the tools available to tackle those challenges are improving.

In 2024, the RNA research community is likely to see major leaps forward in the advancement of all RNA modalities throughout all phases of development.

Emerging RNA therapeutics

The three main RNA modalities that are under way differ in their size and shape. Unlike mRNA, which is linear, circRNA is a closed loop of RNA. Its rolling shape offers higher resistance to nucleases and the potential of increased translation to proteins compared with mRNA.

Furthermore, the human body makes circRNA naturally — a process that is known to be dysregulated in certain disorders and diseases. Recent research has demonstrated a link between the dysregulation of 55 circRNAs and some neurological and psychiatric disorders, including schizophrenia and bipolar disorder.1

The researchers found most of the 22 dysregulated circRNAs in schizophrenia and the 33 in bipolar disorder were downregulated. It is possible, then, that circRNA-based medicines could prove useful as replacement therapies. 

Improving the analysis of RNA medicines: what to expect in 2024

saRNA and srRNA encode for non-structural proteins that are derived from viruses, allowing the RNA to replicate itself using the cell’s machinery after administration.

By contrast, the linear mRNA used in the initially approved COVID-19 vaccines is not self-replicating. The ability of saRNA to self-replicate could help to prolong its effects, reducing the frequency of dosing for the treatment of diseases.

Another benefit could be the reduction of production timelines and costs by generating a therapeutic or vaccine with a much lower required amount per dose than what’s currently required for mRNA.2

In addition, lower required doses to achieve therapeutic effects widen the possible indications for saRNA and srRNA compared with mRNA because of potentially reduced toxicity.

An saRNA vaccine against COVID was approved in Japan late last year and there are several saRNAs to treat cancer, genetic disorders, neurological diseases and more in different phases of development.

The circRNA pipeline is not as far ahead yet, although several partnerships have been formed to fast-track preclinical research efforts.

Confronting hurdles in IVT RNA development

In early development, RNA scientists encounter several challenges when analysing drug candidates. For instance, some big challenges centre around assessing the integrity of the entire molecule, as well as the 5’ cap and 3’ poly-A tail critical quality attributes (CQAs). 

mRNA therapeutics and vaccines measure approximately 1-2 megadaltons (MDa), with saRNA being even larger at approximately 5 MDa. Analysing the intact IVT RNA molecule and separating impurities can be a difficult ask for some analytical methodologies.

Although circRNAs do not contain 5’ cap and 3’ poly-A tails, they are crucial to ensure the stability and translation efficiency of mRNAs and saRNAs. The poly-A tail usually consists of approximately 100–150 adenosine nucleosides and is heterogenous.

Determining its length and distribution accurately poses an analytical challenge, especially considering the lack of commercially available standards. 

Furthermore, the desired 5’ cap structure only differs 1–2 methyl groups from non-mature cap versions. This translates to a difference of 30,000 to 300,000 times versus the intact IVT RNA.

Assessing such subtle differences is not a simple analytical task for any method. To put that into perspective, traditional drugs are more uniform and significantly smaller: aspirin (acetylsalicylic acid) has a mass of approximately 180 Da.

Improving the analysis of RNA medicines: what to expect in 2024

Analysing integrity, purity and other CQAs is difficult; not only because of the overall magnitude and size differences, but also because of the heterogeneity of RNA species present in the sample.

Typically, scientists use liquid chromatography (LC) or gel electrophoresis to study the intact molecule. To assess the 5’ and 3’ ends, a common approach is to enzymatically cut them off and analyse them using standalone LC or LC coupled to mass spectrometry (LC-MS). 

The US Food and Drug Administration (FDA) and other regulatory agencies require impurities to be separated from the drug to determine its purity as part of the release criteria. This is a difficult proposition for IVT RNAs, which are both large and heterogenous and therefore demand very high-resolving analytical techniques. 

In the case of circRNAs, impurities might not differ in the number of nucleosides at all, but only in topology. This poses challenges to the scientific community in terms of achieving drug approval for newly emerging modalities … for which standards for analysis have yet to be established.

However, the US Pharmacopeia (USP) is very aware of the need for nucleic acid standards to support the development and approval of new drug candidates and some gaps might be closed in 2024.

Another major research focus pertains to the delivery vehicle of IVT RNAs.

The most widely used delivery method is to enclose them in lipid nanoparticles (LNPs). Analysing LNPs is vital to optimising stability — including protecting the drug cargo from degradation — and the delivery to the target cells.

Coupling lipids with proteins that bind to the target cells is one approach being developed to improve delivery and cellular uptake. It is also important to understand encapsulation efficiency, which is what percentage of LNPs are filled with the genetic cargo and are empty of or filled with degraded RNA.

A method employing a fluorescent dye is frequently used to quantify encapsulation efficiencies, but it is far from perfect, and demand for better LNP analysis techniques is high.

Emerging analysis solutions

One analytical technology that is highly relevant in the RNA field is capillary electrophoresis (CE). The electric field that is applied results in the migration of RNA molecules through the capillary and causes separation based on their size and charge.

Compared with LC methods, CE-based methods show significantly better resolution of large RNA species and can be used to determine purity and integrity.

CE marks a significant improvement compared with traditional slab gel electrophoresis in terms of resolution, reproducibility and automation capability; plus, state-of-the art instrumentation allows for temperature control to prevent the formation of secondary structures. The technology is suitable for linear mRNA, saRNA species and circRNA.

In a 2023 study, multicapillary gel electrophoresis was coupled with a laser-induced fluorescence detector (CGE-LIF) to separate circRNAs from their linear precursors.

The showcased workflow achieved the separation and purity assessment of the circular product and revealed an additional RNA species that might be linked to a partially nicked form. The method was able to reproducibly detect impurities that were present at very low levels (0.1%) relative to the main peak.3 

The same CGE-LIF workflow can also be used to analyse LNP encapsulation. Another study compared the workflow with the traditionally used fluorescent dye test and found that CGE-LIF determined comparable encapsulation efficiency results while allowing for simultaneous assessment of the integrity of the RNA cargo.4

The ability to analyse purity, integrity and LNP encapsulation in one workflow can improve results and efficiencies in the analysis process.

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This year will no doubt bring continued improvements in the technologies and workflows used to investigate RNA therapeutics.

As these analysis methods improve, we hope that researchers will be able to develop larger both saRNA and circRNA molecules, and mRNA constructs, widening applications in the treatment and prevention of diseases. 

Increasing the efficiency of RNA analysis workflows can lower development costs and help to bring drugs to patients faster. More temperature stable IVT LNP formulations will facilitate global distribution.

Improvements in delivery and administration could usher in RNA drugs that can be inhaled or delivered via skin patches containing microneedles.

These advances could offer significant benefits, such as the reduced need for trained personnel to administer injections that will make RNA drugs and vaccines more widely accessible and affordable for healthcare systems worldwide — a crucial step toward quicker responses in future pandemic scenarios.

This is an exciting time in the evolution of IVT RNA. From vaccines to therapeutics and medicine overall, all these advances will benefit patients and healthcare systems.


  1. E. Mahmoudi, et al., “Dysregulation of circRNA Expression in the Peripheral Blood of Individuals with Schizophrenia and Bipolar Disorder,” J. Mol. Med. 99, 981–991 (2021).
  2. Y. Liu, Y. Li and Q. Hu, “Advances in saRNA Vaccine Research Against Emerging/Re-Emerging Viruses,” Vaccines (Basel) 11(7), 1142 (2023).

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