(147f) Novel Adsorptive Membranes for mRNA Capture for Vaccine Manufacture

The highlight of the Covid-19 pandemic, among others, has been the enormous potential of mRNA vaccine as therapeutics. Currently, vaccines for cancer, HIV, Nipah virus and therapeutics for auto-immune disorders based on mRNA technology are also under different phases of clinical trials. However, a robust downstream processing system that is fast, scalable and suitable for these sticky high molecular weight nucleic acids is required for greater accessibility of this technology. Continuous vaccine production via purification of mRNA will speed-up manufacturing and reduce costs of production. 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, t_R, (ii) allow continuous processing, (iii) retain a higher percentage of folded mRNA due to the reduced treatment residence time, t_R, and (iv) significantly reduce the “footprint” of the equipment. This work introduces a membrane-based capture of mRNA with surface functionalized regenerated cellulose (RC) flat sheet membranes.

The work focuses on using the SET-LRP technique of surface grafting to introduce functional groups on the surface of RC membranes that can bind mRNA from a solution. A surface binding estimation is first carried out based on the hydrodynamic radii of the family of secondary structures of mRNA. This calculation determines the maximum adsorption capacity from a monolayer of surface probes. The RC membranes are first modified by addition of BIBB(α-Bromoisobutyryl bromide) to introduce Br sites. This is followed by either a one-step or a two-step grafting technique for surface modification. The different parameters influencing degree of grafting, such as, BIBB concentration, polymerization time, effect of solvent, monomer to solvent ratios are optimized with the goal of maximum binding of mRNA on the bottle brush layers of modified RC membrane. The modifications are carried out in a 96-well-plate format for a high throughput screening of characteristics. The modified surfaces are characterized by ATR-FTIR and surface fluorescence for and the density of functionalization is correlated with the reaction conditions.

For performance characteristics of the membranes, pure water and buffer flux data is recorded with a 13mm Swinney filtration setup for both unmodified and modified RC membranes. This is then compared with the filtration data obtained from mRNA capture with the same buffer conditions. The elution of the captured mRNA under different buffer conditions is also investigated and the dynamic binding capacity data is recorded. The challenge here is to adapt the surface grafting technique with hollow fiber RC membranes with much larger surface areas that would ensure a greater amount of capture per unit volume. A complete purification scheme involving both capture and polishing of mRNA is developed. Resolving the bottleneck in downstream processing in the manufacture of mRNA therapeutics with novel membranes for effective, fast purification is the ultimate aim of the project.