A Platform Approach to mRNA Purification by Tangential Flow Filtration: Application to a Pentavalent Norovirus Vaccine

Background/Objectives: Multivalent mRNA vaccines are being evaluated for prevention of norovirus infection caused by multiple circulating strains. Platform purification processes may accelerate development and reduce manufacturing complexity by enabling multiple mRNA constructs to be processed using a common downstream workflow. This study aimed to develop a high-yielding tangential flow filtration (TFF) process for a pentavalent norovirus vaccine comprising five VP1-encoding mRNA constructs from genotypes GI.1, GII.2, GII.3, GII.4 and GII.6. Methods: TFF studies evaluated membrane MWCOs, transmembrane pressures, shear rates, mRNA loadings, and scale-up. Process performance was assessed based on mRNA recovery, integrity, functionality, and apparent hydrodynamic size. Results: Although the VP1 constructs had similar molecular weights (590–610 kDa), initial studies using 100 kDa membranes revealed construct-dependent losses due to permeation of full-length mRNA. Apparent hydrodynamic size was associated with observed membrane sieving behavior, suggesting that transcript-specific structural properties may influence membrane transmission. Reducing the membrane MWCO from 100 to 50 kDa eliminated detectable mRNA permeation while maintaining membrane performance. A shear rate of 4000 s−1 supported higher mRNA loading without compromising recovery or integrity. The resulting 50 kDa process was successfully scaled from 40 cm2 to 1600 cm2 and achieved product yields exceeding 95% across all five VP1 mRNA constructs. Conclusions: These findings support a framework for developing standardized TFF conditions for multivalent mRNA vaccines. The results indicate that transcript molecular weight and nominal membrane MWCO alone may not predict mRNA retention and demonstrate the applicability of a common TFF process across five VP1 mRNA constructs. The proposed influence of RNA conformation on membrane transmission requires confirmation using orthogonal structural methods.