Salt gradient and ion-pair mediated anion exchange of intact messenger ribonucleic acids

Abstract

Small molecule drugs and protein-based therapeutics are widely used to treat various diseases, yet even more aspects of human health can be addressed through new modalities, like messenger RNA (mRNA). The complex nature and chemical structure of an mRNA molecule will require the development of new analytical methods. In this work, we propose the use of ion exchange and relatively mild elution conditions as the basis for alternative analytical methods. We have found that a gradient of weak ion-pairing cations can produce intriguing AEX separations of mRNA. This so-called ion pairing anion exchange “IPAX” method provides different recovery and selectivity effects compared to classical sodium chloride salt gradients. We have also shown there to be value in combining the use of an ion-pairing agent as a mobile phase additive along with the implementation of a classical salt gradient. It might be most appropriate to refer to this separation technique as an “ion-pairing mediated salt gradient”. The column temperature has been found to be a very important factor that should be applied to test for effects on various chromatographic values and to learn about an mRNA molecule while confirming its optimal separation conditions. In a classical salt gradient separation, relatively low temperatures (T = 30–40 °C) resulted in better recoveries while in the IPAX separation, higher temperatures up to 60 °C seemed to be more beneficial. An ion-pairing mediated salt gradient appeared to be best matched to temperatures between 30 and 45 °C. Plotting van't Hoff data has also elucidated new information and that a classical salt gradient seems to be an entropy driven process, while the IPAX separation seems to be more complex. It exhibits an entropy driven process at lower temperatures and then an enthalpy controlled one at higher temperatures.