Christine Vo

Limited chromatographic resolution of large nucleic acids remains a major bottleneck in the development of high-throughput analytical platforms for mRNA therapeutics. Here, we report a pronounced on-off elution behavior of oligonucleotides and mRNA under strong ion-exchange (IEX) conditions, using a normalized linear ionic strength model. This discovery fundamentally redefines solvent-strength scaling in IEX and enables rapid separations of oligonucleotides and mRNA on ultra-short columns. Leveraging this on-off elution behavior, two complementary high-throughput IEX methods were developed using 5 cm columns under a unified platform (pH 8, 75 °C, lithium perchlorate as the mobile-phase additive). A 6.5-minute gradient achieved baseline separation and quantification of non-poly(A)- and poly(A)-mRNA species for both eGFP and Fluc mRNA, representing the first reported baseline chromatographic separation of intact non-poly(A)- and poly(A)-mRNA. To resolve poly(A) length variants within the oligonucleotide sequence range, a non-linear logarithmic gradient achieved separation of 20-120 nt poly(A) ladders within 4 minutes, enabling high-throughput poly(A) tail length determination. Following one-step RNase T1 digestion, poly(A) tail lengths of 117 nt and 118 nt were determined for eGFP and Fluc mRNA, respectively, in agreement with theoretical values. Collectively, this work establishes on-off elution behavior as a defining retention regime in IEX and demonstrates it as a powerful strategy for high-throughput characterization of mRNA poly(A) content and tail length, with strong potential to support process development and quality control of mRNA therapeutics.