(93d) Advanced Purification of mRNA with Primary Amines for the Removal of Double Stranded Species

Following the accelerated development of mRNA as a viable tool in disease treatment, catalyzed by the COVID-19 pandemic, we have entered a new era of vaccinology. Much of the success of mRNA therapeutics lies in its scalable in-vitro (cell free) synthesis; however, inherent inefficiencies in this biological process often result in the production of small quantities of unwanted byproducts. As the biological understanding of the effects of these impurities on patients increases, it is becoming clear that double stranded species, notably dsRNA, play a significant role in the immunogenicity of mRNA therapeutics. As dsRNA often shares similar physiochemical properties with target ss-mRNA and cannot be entirely removed by established oligo-dT capture methods, addressing the existence of these species poses a significant separation challenge at scale. This work describes a convective-based primary amine membrane separation capable of balancing anion exchange as well as hydrogen bonding interactions. Ultimately, this approach yields a scalable and productive platform designed to improve the removal of double stranded species.

In ongoing efforts to advance the utilization of modified regenerated cellulose (RC) membranes in mRNA purification, this presentation will explore the modification and characterization of the membrane surface, alongside the experimental quantification and optimization of membrane efficacy concerning dynamic mRNA capture and separation from process impurities. A membrane surface modification method, known as single electron transfer – living radical polymerization (SET-LRP) will be described, resulting in primary amine concentrations up to 26 mol/m². Additionally, a high-performance liquid chromatography (HPLC) adaptable method will be outlined, resulting in Fluc-mRNA DBC_10%of 1.4 mg/mL, with a recovery of bound single stranded nucleic acids up to 100%. Various optimization studies will be detailed to enhance the recovery of bound nucleic acids and the separation capabilities of the membranes for various double-stranded species. Ultimately, an investigation into the reusability and scalability of the membrane devices will shed light on the industrial promise of membrane technology for mRNA-based applications.

Overall, the findings suggest that the modified RC membranes offer a superior alternative to existing industrial removal methods for double stranded species. The membrane system harnesses a convective nature to achieve high mRNA recovery and effectively remove double-stranded species in a scalable format. With its high productivity, the system presents attractive process economics and holds the potential to mitigate the immunogenicity of mRNA-based therapeutics.