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Analysis of mRNA multimerisation (aggregation) using non-denaturing ion-pair reversed-phase liquid chromatography.

Alexandra L J Webb¹, Emma N Welbourne¹, Thomas C Minshull¹

¹School of Chemical, Materials and Biological Engineering, University of Sheffield, Sheffield S1 3JD, UK.

Journal of Chromatography. A

|January 13, 2026

Summary

A new ion-pair reversed-phase HPLC method effectively detects mRNA multimers, crucial impurities impacting mRNA medicine safety and efficacy. This technique offers significant advantages over current methods for analyzing mRNA aggregates.

Keywords:

Ion-pair reversed-phase HPLC Mass photometry mRNA aggregates mRNA medicines mRNA multimers

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iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution

iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution

Published on: April 30, 2011

Area of Science:

Biochemistry
Analytical Chemistry
Pharmaceutical Sciences

Background:

Messenger RNA (mRNA) medicines offer broad therapeutic potential but can contain impurities like mRNA multimers (aggregates).
These mRNA aggregates can compromise the safety and efficacy of mRNA-based therapies.
Current analytical methods often fail to detect these critical mRNA multimers.

Purpose of the Study:

To develop and validate a non-denaturing method for analyzing mRNA multimers.
To characterize the formation and properties of mRNA multimers.
To establish a quantitative method for assessing mRNA aggregation.

Main Methods:

Development of ion-pair reversed-phase high-performance liquid chromatography (IP-RP HPLC) under non-denaturing conditions.
Utilisation of Mg²⁺ in the mobile phase to stabilize mRNA higher-order structures and facilitate multimer separation.
Mass photometry analysis for characterization of purified mRNA multimers.

Main Results:

Successfully resolved mRNA monomers from dimers/multimers across various sequences and lengths using non-denaturing IP-RP HPLC.
Demonstrated that mRNA multimer abundance is concentration-dependent.
Quantified the dissociation constant (K_d) for eGFP mRNA dimer formation as 82.93 nM.

Conclusions:

Non-denaturing IP-RP HPLC provides a significant advancement for mRNA multimer analysis compared to existing methods.
This high-throughput method enables detailed studies on mRNA multimer stability and factors influencing aggregation.
The findings are vital for ensuring the quality and safety of mRNA medicines.