Development of an Oligo(dT) chromatography step for purifying in vitro transcription generated mRNA

Oligo(dT) chromatography can be used for the selective capture of mRNA through the A to T base pairing between the PolyA tail of the mRNA and the dT ligand of the resin. Oligo(dT) has been used to improve mRNA integrity and remove residual impurities from the In Vitro Transcription (IVT) reaction such as residual DNA template and dsRNA. These impurities are present after capture because they are either interacting with the resin or the mRNA and those interactions are most likely ionic, metal complexation, and/or hydrogen bonding. To improve the reduction of these impurities while maintaining mRNA integrity, development of an intermediate wash step was performed by screening different wash solutions over a range of salt concentrations by implementing high throughput plate screens and robocolumns. The results of the high throughput screens were then confirmed at bench scale to determine the optimal wash for impurity removal. Additionally, monolith Oligo(dT) columns were studied to evaluate their impact on mRNA binding capacity, processing time, and impurity removal compared to resin-based Oligo(dT).