Accurate evaluation of mRNA capping efficiency

Accurate analysis of mRNA capping efficiency is essential for ensuring the quality, stability, and therapeutic performance of modern mRNA-based medicines. This Application Note presents an optimised ion-pair reversed-phase liquid chromatography (IP-RP LC) workflow using the YMC Accura Triart Bio C18 column for efficient analysis of capped and uncapped in vitro transcribed (IVT) mRNA.

Challenges in analysis of intact mRNA

Messenger RNA therapeutics require precise quality control because the 5’ cap structure strongly influences translation efficiency, transcript stability, and immune response modulation. While Cap-0 contains a 7-methylguanosine moiety, Cap-1 includes an additional methyl group that reduces unwanted immune activation, making it the preferred structure for modern mRNA therapeutics. However, enzymatic capping can generate uncapped or partially capped impurities that negatively affect product quality and efficacy.

Direct chromatographic analysis of intact mRNA is challenging due to the large molecular size. Therefore, capping efficiency is commonly assessed after RNase H-mediated fragmentation, which generates short 5’-terminal oligonucleotides suitable for chromatographic analysis. Separating structurally similar capped and uncapped species requires carefully optimised chromatographic conditions.

Optimised IP-RP LC workflow with YMC Accura Triart Bio C18

YMC developed an optimised IP-RP LC method using the wide-pore YMC Accura Triart Bio C18 column with bioinert hardware. The bioinert-coated YMC Accura hardware reduces analyte-metal interactions and improves reproducibility, while the stationary phase enables efficient mass transfer and high-resolution separations. The optimised conditions enabled clear separation of capped and uncapped IVT mRNA species differing by only one nucleotide.

Sensitive LC–MS detection of capping variants and impurities

The method was also successfully applied to LC–MS analysis of Cap-1 modified mRNA species, enabling differentiation between Cap-1, Cap-0, partially methylated intermediates, 5’-diphosphate species, and uncapped impurities, supporting confident quality control of mRNA therapeutics.

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