(505d) Purifying mRNA Therapeutics: We Need a Paradigm Shift

Increasing clinical trials of single stranded mRNA (ssRNA) therapeutics highlight the urgent need to develop efficient, scalable, and economical purification methods. Current diffusion-driven, resin-based purification techniques constrict productivity and rely on expensive oligo-dT ligands for facilitating hybridization with target ssRNA poly-A tails. Hence there is now an urgent need to develop a less costly, fast and efficient affinity mRNA capture step with the following requirements: High recovery, high ligand utilization, high productivity and capacity reduced residence times for maintenance of RNA integrity, small equipment footprint, modularity and efficient removal of product and process impurities such as genomic DNA, T7-RNA polymerase, double-stranded, capless and fragments of immunogenic mRNA.

Minimizing the immunogenicity of process- and product-related impurities resulting from in vitro transcription production of ssRNA biologics is crucial. Double stranded RNA (dsRNA), which can vary in length and structure, presents a particular challenge. While the immune system can tolerate low levels of dsRNA in single-dose vaccinations, dsRNA molecules longer than forty base pairs can elicit an innate immune response, necessitating their removal to ensure the safety and efficacy of ssRNA therapeutics. For multiple and larger doses for cancer and other diseases, immunogenic dsRNA cannot be tolerated.

Purification of ssRNA vaccines has saved millions of lives. However, since all purification processes are not 100% efficient, small amounts of immunogenic compounds do leak through with purified ssRNA product causing significant unpleasant immunogenetic responses but not death. Here, we (i) show that, with oligo-dT ligands, convective membrane and monolith processes far outperform chromatographic diffusive processes¹, (ii) discover, graft and test our patented selective microporous affinity peptide membranes that selectively bind to ssRNA or dsRNA², and (iii) present the first details and results from our patented cost-effective ligand-less multimodal surface-modified approach to purify ssRNA from dsRNA³. See Fig. 1.

References

(1) Banik, R.; Neuman, T. G.; Hao, Z.; Al Sharabati M.; Zhao, W.; Anderson., D. G.; Przybycien, T.; Kilduff, J.; Belfort, G. Convection Rather than Diffusion for Fast Efficient mRNA Vaccine Purification. Separation and Purification Tech. 2025, 354.

(2) Hu, M.; Neuman, T. G.; Bai, Y.; Belfort, G.; Karande, P. Discovery and Mechanistic Investigations of RNA-Binding Peptides for Therapeutics Development. submitted 2025.

(3) Neuman, T. G.; Banik, R.; Karla, S.; Hu, M.; Hartin, A.; Karande, P.; Kilduff, J.; Belfort, G. Tuning hydrogen bonds and electrostatics with convection for purifying mRNA: A paradigm shift. Scientific Reports 2025, in press.