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

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 utilized high-resolution 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 stabilizes 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 and multimers was demonstrated for a range of mRNA sequences and lengths and was shown to be concentration dependent. Validation of mRNA multimers was performed using mass photometry analysis following the purification of mRNA monomer and dimer/multimer peaks using IP-RP HPLC. The ability to resolve mRNA monomers from mRNA dimers with short chromatographic run times, perform analysis across a wide range of temperatures and concentrations of mRNA combined with the ability to collect the mRNA monomers and dimers/multimers for further downstream analysis 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.