Exploratory study of ion pair liquid chromatography separation methods and sample preparation procedures – for quantification of amount 5’capped and uncapped mRNA

Abstract: Using ion pair chromatography, separation and analysis of shorter mRNA fragments was possible. The aim of the study was to develop a separation method for quantitation of 5’capped versus uncapped mRNA. Cleavage of mRNA was performed by complementary probes and the enzyme RNase H which created shorter mRNA fragments containing the 5’ end. During this study, several separation methods have been tested where the amount organic solvent proved to be a crucial factor for elution of the fragments. To identify the peaks during the method development, LC-MS had to be used which resulted in an adjustment of the methods parameters. With the mass spectrometric analyses could a capped fragment as well as a capped fragment containing an extra guanine nucleotide be identified, however, there was no presence of the uncapped fragment. Enzymatic protocols have been investigated where the purpose was to create uncapped RNA fragments to be used as a reference. A minor validation of the final separation method has been carried out where the generated data can be used to establish acceptable system suitability test criteria for separation methods when analyzing mRNA drug products.