(427g) Development of an Integrated Continuous mRNA Precipitation-Based Purification Process

In recent years, mRNA-based therapeutics, including mRNA vaccines, have emerged as cutting-edge technologies for treating various diseases, ranging from cancer and gene therapy to cardiovascular and autoimmune conditions. Current downstream processing relies on chromatography strategies, such as size exclusion chromatography, ion pair reverse-phase chromatography, ion exchange chromatography, or affinity-based separation, coupled with tangential flow filtration. However, purification remains challenging due to working conditions that can lead to denaturation, high costs, limited scalability, or gel formation and fouling during filtration steps. These extensive intermediate steps significantly impact overall yield and RNA integrity, thus influencing final production costs. Therefore, the need for innovation in manufacturing processing is crucial with the advent of such a new therapeutic modality.

In recent years, precipitation has become an interesting, flexible, easy-scalable, and cost-effective alternative to chromatography systems for purification of therapeutic products such as monoclonal antibodies. purifying therapeutic products, such as monoclonal antibodies. Precipitation aligns with the current trend of continuous manufacturing and sustainability, as it can be performed in a fully continuous mode, reducing energy and material consumption. On the laboratory scale, precipitation serves as a straightforward and cost-effective method for concentrating RNA, involving salts like ammonium acetate or lithium chloride. However, these methods do not allow for the removal of impurities, such as aberrant mRNA, mRNA fragments, or dsRNA, and the scalability of such processes still needs to be addressed. We will present our integrated and continuous manufacturing process for mRNA production and purification. We will present our work on developing and implementing novel methods for continuous mRNA precipitation-based purification. These methods include combining various precipitating agents, such as PEG and different cations, and following precipitation with continuous flow filtration for both concentration and washing. This system is integrated at the benchtop scale for the continuous purification of mRNA.