Novel Mobile Phase to Control Charge States and Metal Adducts in the LC/MS for mRNA Characterization Assays

[Image: see text] Mass spectrometry is a widely used tool in the characterization of oligonucleotides. This analysis can be challenging due to the large number of possible charge states of oligonucleotides, which can limit the sensitivity of the assay, along with the propensity of oligonucleotides to readily form adducts with free alkali metals. To reduce the adduct formation, oligonucleotides are typically purified with desalting columns prior to analysis. We have developed a mobile phase that gives superior reduction in charge states and adduct formation compared to previously reported methods and, more importantly, obviates the requirement of desalting samples prior to mass spectrometric analysis, significantly decreasing the sample preparation time and amount of RNA required for analysis. We have applied this mobile phase to develop methods to quantify the 5′-capping efficiency and to characterize the polyadenosine (poly(A)) tail of mRNA synthesized in vitro: two critical quality attributes of mRNA therapeutics. Through this, we were able to demonstrate RNA that was co-transcriptionally capped to have capping efficiency equivalent (the percent total molecules that contain a cap) to other reports in the literature using materials that were generated using the same synthesis procedure. Furthermore, by using a mobile phase mixture comprised of hexafluoroisopropanol, triethylammonium acetate, triethylamine, and ethanol, we were able to determine the size distribution of the poly(A) tail in various mRNA samples from DNA templates that ranged from 50 to 150 nt poly(A) and verify that distribution with commercially available RNA standards, successfully demonstrating that this mobile phase composition could be used for characterization assays for both mRNA caps and tails.