Novel two-stage tandem chromatography approach for scalable and efficient purification of ssRNA from mRNA production impurities

The increasing demand for mRNA-based vaccines and therapeutics has necessitated the development of more efficient purification methods for mRNA produced via in vitro transcription (IVT). IVT typically yields a mixture of single-stranded mRNA (ssRNA), truncated mRNA, double-stranded RNA (dsRNA), and other impurities. Current methods like oligo d(T) affinity chromatography, which binds the poly(A) tail of mRNA, have limitations, as they fail to fully remove dsRNA that contains exposed single-stranded regions. Additionally, methods such as IP-RP chromatography are not suitable for large-scale production due to their long processing time and their reliance on organic solvents like acetonitrile, which is both toxic and costly.
We present a novel two-step tandem chromatography approach that effectively removes contaminants such as rNTPs, DNA, proteins, dsRNA and fragmented ssRNA from the IVT reaction mixture, without using organic solvents. The first step uses hydrogen bonding pyrophosphate displacement chromatography, which leverages the differential hydrogen bonding capacity of contaminants. This step removes dsRNA, proteins, and other impurities. The second step employs oligo d(T) affinity chromatography, which further purifies the target mRNA by removing truncated mRNA fragments and facilitates removal of pyrophosphate. The process can achieve 65% removal of dsRNA generated by IVT, and above 95% fragment purity at laboratory scale. We also identified mobile phase modifiers that enhance the binding capacity of the hydrogen bonding monolith, specifically to nucleic acids, allowing for higher yields without increasing process time or complexity. Both chromatography steps are performed using commercially available monoliths, which support flow rates up to 10 mL/min and shorter retention times compared to traditional beaded resins, making the process suitable for industrial-scale production.