Structure-performance analysis of oligo(dT) resin affinity chromatography for RNA separations

• Poly(A) is bound in shallow pores close to the exterior of beaded oligo(dT) resins. • Strong residence time dependency of DBC indicates diffusive mass transport of the feed to binding sites. • Residence time dependency of DBC is influenced by resin surface morphology and ligand density. • DBC positively correlates with higher ligand density, but the mass transport speed of poly(A) needs consideration. mRNA is an emerging drug modality with high potential as both therapeutic and prophylactic products. The rising investment in this technology can be attributed to its versatility and rapid development of new drug candidates. Oligo deoxythymidine (oligo(dT)) ligand chemistries are of industrial relevance in chromatographic purification of mRNA, however, beaded oligo(dT) resins struggle with low dynamic binding capacity (DBC). To aid understanding of these beaded oligo(dT) resins, a structural analysis was conducted, followed by a characterisation of poly-adenine RNA binding behaviours. The aim establishing a structure-performance relationship to inform future resin design. To characterise structural properties of resins, Brunauer-Emmett-Teller analysis first determined the surface area of three resins, ranging from 13–18 m 2 .g −1 . Mercury intrusion porosimetry characterised pore size distribution and scanning electron micrographs were produced of resin surfaces and interiors from bead cross-sections, observing modal pore diameters of 298–526 nm. To measure RNA binding performance, we tested binding of a 2100–10,000 nucleotide poly-adenine over 20 min and characterised material binding. Material predominantly collected on the bead exterior, but gradual penetration occurred into the internal pore network. Breakthrough experiments connected these results to trends in DBC. A 257 % DBC 10 increase was observed from 1–10 min residence time, highlighting the importance of diffusive transport in these beaded resins. We also compared mass transfer behaviours between two RNAs with median lengths of ∼500 and ∼5000 nucleotides, observing significant differences in diffusive transport speed, with larger RNA entering the bead interior more slowly. With this work we aim to inform future resin design for mRNA purification.