Publications

Since the clinical trials for the first COVID-19 vaccines in 2020, interest in RNA-based therapeutics has grown rapidly, with promising applications in vaccines, oncology, and gene therapy. This surge has created a strong demand for scalable, cost-effective, and robust manufacturing platforms for messenger RNA. However, current mRNA purification largely relies on batch-wise chromatography and tangential flow filtration, which face limitations in scalability, cost, and compatibility with continuous production. Chromatographic techniques often require harsh conditions, such as high pH, salt, or organic solvents, that may compromise mRNA stability. Additionally, extensive sample conditioning (e.g., dilution, heating) is typically required to reduce aggregation and facilitate column loading, further hindering continuous operation. To address these challenges, a fully continuous precipitation-based method for mRNA purification is developed. The process consists of an optimized precipitation step using PEG6000 and NaCl in a tubular reactor, followed by two continuous TFF stages for washing and buffer exchange. The overall process achieves yields of 92% and purities of 95%, with no detectable double-stranded RNA formation, residual proteins, fragmentation, or aggregates. Compared to traditional approaches, this method achieves higher yields and purities while offering enhanced process robustness and integration potential. The final mRNA product can be directly encapsulated into lipid nanoparticles without further conditioning, with no observed degradation or aggregation. This platform offers a scalable, flexible alternative to chromatography, suitable for integration into end-to-end continuous mRNA manufacturing.

Lipid nanoparticles (LNPs) are the leading platform for delivering nucleic acid therapeutics, produced by rapidly mixing lipids in ethanol with nucleic acid cargo in an aqueous buffer. LNP production is often approached with a mixing-focused mindset that reduces the entire self-assembly process to a single step, obscuring the relationship between the process inputs and LNP properties. Here, we present a method for producing mRNA-loaded LNPs, with independent and predictive control over both the size and morphology and without compromising other quality attributes. By decoupling particle design from mixing and formulation changes, this method enables the rational engineering of LNPs with defined properties. The method leverages mixing under high fusogenicity conditions, achieved by modulating the solvent composition, followed by timed postinjection of an aqueous buffer to kinetically arrest LNPs at the desired properties. We demonstrate the method using benchmark LNP formulations in an impinging jet mixer, a state-of-the-art technology for LNP manufacturing. The resulting LNPs exhibit up to an 8-fold increase in in vitro transfection efficacy compared to those produced by the conventional method. In addition, the method facilitates quality control and supports predictive modeling and rational process translation.

The increasing demand for mRNA-based therapeutics requires scalable and cost-effective purification methods. Chromatography-based approaches, such as Oligo-dT affinity chromatography, require multiple processing steps, including buffer exchange and high-temperature treatments, which can lead to mRNA degradation and increased costs. Although precipitation is commonly used at the lab scale, its application in large-scale mRNA purification remains underexplored. In this study, we developed a rapid and efficient precipitation-based method for mRNA purification. The effects of different precipitation conditions, including salt type, co-precipitating agents, and time, were evaluated in terms of recovery yield and purity. Precipitation experiments were conducted for two different in vitro transcription crude materials containing mRNAs of varying lengths and concentrations. The optimal conditions, identified as a combination of NaCl and PEG 6000, achieved recovery yields of 80-93% and purities of 80-83%, with no detectable double-stranded RNA formation or fragmentation. The precipitated mRNAs were successfully transfected into 293T and A549 cells, showing protein expression comparable to commercial and chromatography-purified mRNAs. These findings position PEG/NaCl precipitation as a rapid, scalable, and cost-effective alternative to traditional chromatography methods, significantly reducing process complexity and time for large-scale mRNA production.