Optimizing mRNA Analysis Using Capillary Electrophoresis

Using capillary gel electrophoresis, researchers from the National Institute of Health Sciences in Kanagawa, Japan studied the factors that optimize chain-length distribution analysis of messenger RNA (mRNA). Their findings were published in the Journal of Chromatography A (1).

Messenger RNA (mRNA) is a type of single-stranded RNA created from DNA templates during the transcription process (2). Typically, mRNA is used to carry protein information from DNA in a cell’s nucleus to its cytoplasm, where protein-making machinery reads the mRNAsequence and translates the codons into their corresponding amino acids in a growing protein chain. This type of RNA has been used in various applications, though its use became more frequent with the creation of the coronavirus disease-2019 (Covid-19) vaccines. Regardless of what was used in their creation, tremendous increases in mRNA vaccine research funding were made in the process of combatting Covid-19 (3).

The creation and population of mRNA vaccines has led to active clinical development of mRNA-based modalities, such as vaccines for infectious diseases and cancer, therapeutics for genetic disorders and tissue damage, and Cas9-encoding mRNA for genome editing therapies. These modalities, according to the scientists, allow for rapid designing and manufacturing following the identification of the gene sequence of the target pathogen or protein. Further, these present the advantages of rapid mRNA therapeutic development in short periods, simply by changing sequences once development platforms are established, including mRNA structure, type of modified nucleic acid, delivery carrier, manufacturing method, and formulation technology.

Capillary gel electrophoresis is a technique used for separating macromolecules, such as proteins and nucleic acids, by using a sieving medium made up of a crosslinked gel or an entangled polymer network (4). Unlike standard gel electrophoresis, this approach allows for automation, high separation efficiency, on-capillary detection, anticonvective nature of the capillary, and array dimension (being a multicapillary system). Capillary electrophoresis has been used to examine mRNA chain length, and is believed to be sufficient for separating full-length mRNAs and impurity RNAs (shortmers and longmers) to accurately evaluate mRNA quality. However, the analytical parameters that affect mRNA separation have not been adequately verified.

In this study, the scientists analyzed the analytical parameters of capillary gel electrophoresis. By doing so, they found that the gel concentration, denaturant, preheating treatment, capillary temperature, and fluorescent dye remarkably affect the separation of long-chain-length RNAs. The separation limits of capillary gel electrophoresis analysis were examined under adjusted analytical parameter conditions. RNAs of approximately 4000 nucleotides length and their defective RNAs of ≥200 nucleotides could be effectively separated. Additionally, when RNA separation under the researchers’ adjusted conditions was compared with those under conditions recommended in the United States Pharmacopeia (USP) draft guidelines, their method displayed higher RNA separation for both RNA prepared in this study and that of the approved mRNA vaccine.

The analytical parameters outlined in this study could be vital for optimizing RNA separation resolutions, especially in cases where detailed quality analysis is required, such as for characterizing mRNA therapeutics. The scientists believe their findings will be useful for future efforts to optimize analytical conditions for evaluating the chain-length distribution of RNA using capillary gel electrophoresis.

References

(1) Yamamoto, T.; Yoshida, T.; Uchida, Y.; Ohoka, N.; et al. Analytical Factors for Optimization of Chain-Length Distribution Analysis of mRNA Using Capillary Gel Electrophoresis. J. Chromatogr. A 2025, 1754, 466019. DOI: 10.1016/j.chroma.2025.466019

(2) Messenger RNA (MRNA). National Human Genome Research Institute 2025. https://www.genome.gov/genetics-glossary/Messenger-RNA-mRNA (accessed 2025-6-16)

(3) Beyrer, C. The Long History of mRNA Vaccines. Johns Hopkins University 2021. https://publichealth.jhu.edu/2021/the-long-history-of-mrna-vaccines (accessed 2025-6-16)

(4) Capillary Gas Electrophoresis. ScienceDirect 2000. http://sciencedirect.com/topics/chemistry/capillary-gel-electrophoresis (accessed 2025-6-16)