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

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.