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

mRNA-based technology has emerged as a new class of medicines with a wide range of applications, including viral vaccines, cancer vaccines, and therapeutics for the treatment of metabolic diseases and cardiovascular conditions. Impurities, including double-stranded RNA (dsRNA), mRNA fragments, and mRNA multimers (aggregates) that result from the manufacturing of mRNA, as well as from subsequent purification, formulation, and storage, can potentially impact the safety and efficacy of mRNA medicines. mRNA higher-order structures and mRNA multimers (aggregates) can affect translational efficiency and also impact the efficiency of formulation into lipid nanoparticles. mRNA purity is typically analysed using denaturing or partially denaturing methods, precluding the detection of mRNA multimers (aggregates). In this study, we developed and utilised ion-pair reversed-phase HPLC (IP-RP HPLC) under non-denaturing conditions to analyse mRNA multimers. The inclusion of 1 mM Mg²⁺ in the mobile phase stabilises mRNA higher-order structures, RNA:RNA interactions, and the formation of mRNA dimers/multimers, which can be readily separated from the mRNA monomers. The ability to resolve mRNA monomers from mRNA dimers/multimers was demonstrated for a range of mRNA sequences and lengths. Moreover, we have shown that the relative abundance of mRNA dimers/multimers is concentration dependent. Using the relative percentage of dimer vs concentration of monomer, we were able to determine that the K_d of the interaction between two eGFP mRNA monomers was 82.93 nM. Characterisation and sizing of the mRNA multimers was performed using mass photometry analysis following the purification of mRNA monomer and dimer/multimer peaks using IP-RP HPLC. Thus, non-denaturing IP-RP demonstrates significant advantages over current approaches for the analysis of mRNA multimers (aggregates). The high-throughput, temperature-dependent profiling of mRNA multimerisation using IP-RP HPLC will enable further comparative studies on the stability of mRNA multimers and provide important insights into potential factors influencing mRNA multimerisation.