Separation of long-stranded RNAs by RP-HPLC using an octadecyl-based column with super-wide pores

Messenger ribonucleic acids (mRNAs) have been used in vaccines for various diseases and are attracting attention as a new pharmaceutical paradigm. The purification of mRNAs is necessary because various impurities, such as template DNAs and transcription enzymes, remain in the crude product after mRNA synthesis. Among the various purification methods, reversed-phase high-performance liquid chromatography (RP-HPLC) is currently attracting attention. Herein, we optimized the pore size of the packing materials, the mobile phase composition, and the temperature of the process; we also evaluated changes in the separation patterns of RNA strands of various lengths via RP-HPLC. Additionally, single-stranded (50-1000 nucleotides in length) and double-stranded (80-500 base pairs in length) RNAs were separated while their non-denatured states were maintained by performing the analysis at 60 °C using triethylammonium acetate as the mobile phase and octadecyl-based RNA-RP1 with super-wide pores (> 30 nm) as the column. Furthermore, impurities in a long-stranded RNA of several thousand nucleotides synthesized by in vitro transcription were successfully separated using an RNA-RP1 column. The columns used in this study are expected to separate various RNA strands and the impurities contained in them.