Project Summary The goal of this Phase I SBIR project is to develop a novel purification method that is compatible with in vitro transcription (IVT)-based manufacturing of RNA for use as a therapeutic or vaccine. As RNA becomes more commonly used for pharmaceutical products, manufacturing methods must improve their scalability, speed, and cost-effectiveness. There are several pain points in RNA manufacturing. First, current affinity resins have very low capacity —only ~2mg RNA per mL— necessitating large volumes of resin at commercial scale. These resins are priced at over $25,000 per liter, rendering commercial scale production extremely costly. Second, 5’ capping of RNA – essential for in vivo function – and the subsequent removal of any residual uncapped RNA is a major bottleneck for RNA production. Isolere Bio, Inc. specializes in designing innovative purification solutions that combine a protein affinity tag that is genetically fused to an elastin-like polypeptide (ELP). This reagent is capable of temperature or salt-controlled phase separation that enables capture of a specific target regardless of its complexity or the presence of other impurities. We will develop a novel combination of biopolymers, referred to here as IVTag™, that enables simultaneous RNA sequestration into concentrated, pure liquid droplets and methylation of transcripts. Two ELP fusion proteins will be developed: one will contain an RNA-binding motif and the second an O’-methyltransferase (O’-MTase) enzyme, responsible for 5’-capping of RNA. We will then develop platform methods for simultaneous purification of RNA into droplets and high efficiency capping of the sequestered RNA. This Phase I program is designed to demonstrate compatibility of this essential IVT enzyme and complexing it with Isolere’s non-chromatographic purification methods. Key activities of the proposal include synthesizing, purifying, and testing each biopolymer fusions’ functionality and then evaluating their efficacy when applied in tandem and comparing process efficiency to the industry standard method. If successful, additional functionality of the RNA micro-crucible will be developed in Phase II in the form of a T7 polymerase-ELP fusion (for initial transcription of a DNA template) and a Poly-A polymerase-ELP fusion (for adding a 3’ poly-A tail to transcripts). Our vision is to ultimately create a fully continuous RNA manufacturing process in smart droplets. These smart droplets are expected to support a straightforward, scalable, and cost-effective manufacturing workflow with micro-crucibles capable of carrying out a complex series of processing steps. Because the micro- crucible is volume independent, the scale-up difficulties associated with affinity resins are eliminated. In addition, with other applications of Isolere’s technology, including to antibodies and viral vectors, the ELP fusion reagents have proven to be easily manufacturable and re-usable for multiple purification cycles, reduc...