(160d) Novel Adsorptive Membranes for mRNA Capture for Vaccine Manufacture

The highlight of the Covid-19 pandemic, among others, was the enormous potential of mRNA vaccine as a therapeutic. In the rush to manufacture mRNA vaccines and ensure mRNA integrity and high purity, several capital-intensive and time-consuming purification steps were used¹,typically involving costly and time-consuming processes like reverse-phase high-performance liquid chromatography². To manufacture these mRNA products, a robust cost-effective downstream processing platform, that is fast, scalable and suitable for purifying these sticky, labile and high molecular weight nucleic acids, is required. Replacing resin-based chromatography (diffusive, slow and hence long residence time), with membranes (convective, fast and hence short residence time) for purification of mRNA vaccines will (i) reduce the residence times of the recovery process, (ii) allow continuous RNA processing that will speed-up manufacturing and reduce costs of production. (iii) retain a higher percentage of folded mRNA due to the reduced treatment residence time, and (iv) significantly reduce the “footprint” of the equipment. This work introduces a membrane-based capture of mRNA with surface functionalized regenerated cellulose (RC) membranes that has the potential to meet these desirable outcomes.

Our research comprises several steps involving (i) affinity membrane synthesis and characterization (ii) membrane testing with oligo-dA60 and firefly luciferase fluc-mRNA, and (iii) mathematical analysis to describe and guide the research. With respect to synthesis, 12 different olig-dT₆₀ affinity ligands (some with Gs interspersed and of different lengths) were successfully grafted throughout the RC membranes using the single electron transfer–living radical polymerization (SET-LRP) technique. Both a one-step and a two-step method were successful. The modified surfaces were characterized by ATR-FTIR, surface fluorescence and XPS for density of functionalization and correlated with the reaction conditions. Static adsorption isotherms were used to select the best four olig-dT₆₀ affinity ligand binding membranes with both olgo-dA₆₀ and fluc-mRNA. The four selected membranes were then operated in dynamic adsorption mode with both oligo-dA60 and fluc-mRNA in the feed. Standard breakthrough curves were obtained with a maximum of 80% recovery via elution of the bound molecules. To mathematically describe the membrane behavior and distinguish dispersion in the membrane from that in the process tubing and membrane holder, we used two approaches that (i) assessed mixing and pure delay of the system with moment analysis, and (ii) incorporated convection, sorption and dispersion in a transport model. Both approaches described non-ideality of the system well. This on-going research illustrates the potential for mRNA purification using synthetic microporous membranes.

References

(1) Kariko, K.; Muramatsu, H.; Ludwig, J.; Weissman, D. Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA. Nucleic Acids Res 2011, 39 (21), e142. DOI: 10.1093/nar/gkr695.

(2) Crunkhorn, S. Improving mRNA production. Nat Rev Drug Discov 2023, 22 (1), 19. DOI: 10.1038/d41573-022-00200-4.

Acknowledgement

NIIMBL grant with funds from the American Rescue Plan, ARP-08, Jan 10^th, 2022.