(161b) Capturing mRNA with 5’-Cap Selective Peptides Grafted on Membranes

Affinity-based systems have played a pivotal role in advancing downstream processing for mRNA therapeutics, offering high specificity through interactions such as oligo(dT)/polyA hybridization¹. However, currently established downstream processes cannot deplete uncapped mRNA, which can be formed in co-transcriptional capping, in vitro transcription reactions and is potentially immunogenic, translated much less efficiently than capped mRNA. Therefore, approaches to select for correct 5’-capping and separate uncapped mRNA as an important product related impurity, are promising to improve product quality. In this study, we addressed this limitation by immobilizing a selected library of cap-selective peptide ligands onto membranes. The modification process was characterized at each stage of the grafting reactions using FTIR spectroscopy and fluorescence-based assays to quantify ligand density, while reaction conditions were optimized to control surface ligand availability. The peptide-functionalized membranes were evaluated for their cap-binding capabilities. High-throughput screening with two distinct cap analogue structures demonstrated the specificity of the immobilized peptides, and the recovery of capped mRNA was quantified using liquid chromatography-mass spectrometry, enabling precise molecular identification and quantification. Kinetic analysis of capped mRNA binding to the peptide ligands within the membranes was obtained, with binding mechanisms elucidated through modeling of association and dissociation rate constants. This work establishes a novel approach for the selective capture of capped mRNA using peptide-functionalized membranes, offering a promising platform for enhancing the purity and yield with efficiency of mRNA therapeutic production.

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.