Development of integrated and scalable platforms for mrna synthesis, purification, and thermostable mrna-lipid nanoparticle drug formulation

Abstract

August2025School of EngineeringMessenger RNA (mRNA) therapeutics are poised to transform modern medicine, but their widespread adoption is limited by challenges in large-scale manufacturing, impurity removal, and formulation stability. This dissertation presents a series of integrated solutions for the production, purification, and stabilization of mRNA medicines, emphasizing the translation of laboratory innovation into scalable, industrial processes. The thesis work began with the design and synthesis of mRNAs of varying lengths, encoding for concatemeric EGFP proteins, which serve as reference materials for studying the impact of sequence and structure on product quality and delivery. These synthetic mRNAs were thoroughly characterized for integrity, size, and purity using electrophoresis, HPLC, bioanalyzer analyses, and next-generation sequencing (NGS). Following purification, the EGFP mRNAs were encapsulated in lipid nanoparticles (LNPs) using clinically relevant lipid compositions. A comprehensive series of lyophilization protocols were then developed and optimized to produce stable, freeze-dried mRNA-LNP formulations. The physicochemical and functional stability of these lyophilized products was evaluated over extended storage at a range of temperatures, providing new insights into the factors that govern long-term stability of mRNA therapeutics. To address early-stage process impurities, a polyethylene glycol (PEG)-citrate aqueous two-phase system (ATPS) was developed for the rapid and scalable removal of proteins and rNTPs from crude in vitro transcription reactions. The ATPS workflow leverages unique interfacial adsorption properties of mRNA to enable high-yield and gentle separation directly from unprocessed reaction mixtures. This method significantly accelerates the purification process and reduces the burden on downstream chromatographic steps. For final polishing and impurity clearance, a tandem chromatography strategy was established. Hydrogen bonding chromatography was employed as a first step for the efficient removal of double-stranded RNA (dsRNA) and process-related impurities. This was immediately followed by oligo d(T) affinity chromatography, which selectively captures full-length, polyadenylated mRNA. The two-stage process is fully compatible with large-scale manufacturing, provides high product purity, and meets regulatory requirements for clinical-grade mRNA therapeutics. These advancements provide a robust and scalable framework for the manufacturing of high-purity, thermostable mRNA therapeutics. We hope that the resulting workflow not only meets current regulatory and quality demands but also provides a foundation for the broader deployment and global accessibility of next-generation mRNA medicines.Ph