(622b) Advancing mRNA Purification: Peptide Ligand Design for Selective Capture and Purification of 5’-Capped Vs Uncapped mRNA

Messenger RNA (mRNA)-based therapeutics have emerged as a transformative platform for vaccines and gene therapies, necessitating efficient, scalable, and cost-effective purification strategies. A yet to be solved challenge in mRNA manufacturing is the separation of capped mRNA—the therapeutically desired form—from uncapped mRNA byproducts formed during co-transcriptional capping. Uncapped mRNA can be immunogenic, compromise translational efficiency and therapeutic potency. New modalities are therefore in need for efficient cap-specific purification, necessitating the development of novel downstream process strategies with high purity, yield and scalability.

This study addresses this challenge by designing peptide affinity ligands that selectively bind either 5’-capped or uncapped mRNA. These peptides will subsequently be immobilized onto synthetic polymer filtration membranes, enabling the basis for a scalable and high-yield purification process.

Lead peptides were synthesized and screened for binding affinity and selectivity using a high-throughput microarray platform. Peptides exhibiting strong selective binding to capped mRNA with minimal or no binding to uncapped mRNA were identified and further validated via ELISA and microarrays. The top-performing peptides demonstrated up to 100-fold selectivity for capped over uncapped mRNA. Sequence analysis revealed common motifs and amino acid preferences among the strongest binders. Our current efforts are focused on optimizing binding affinity and selectivity of the lead peptides through site-specific mutagenesis and obtaining mechanistic understanding of the selective binding of the peptides using atomic force microscopy and molecular simulations. The confirmed binders will then be grafted onto membranes and tested under process purification conditions. This work establishes a novel peptide-based affinity strategy for highly selective capped mRNA purification, offering a novel mode for the depletion of product-related impurities in mRNA manufacturing.