The goal of this Phase I SBIR project is to develop a novel purification method that is compatible with in vitrotranscription (IVT)-based manufacturing of RNA for use as a therapeutic or vaccine. As RNA becomes morecommonly used for pharmaceutical products, manufacturing methods must improve their scalability, speed, andcost-effectiveness. There are several pain points in RNA manufacturing. First, current affinity resins have verylow capacity -only ~2mg RNA per mL- necessitating large volumes of resin at commercial scale. These resinsare priced at over $25,000 per liter, rendering commercial scale production extremely costly. Second, 5' cappingof RNA - essential for in vivo function - and the subsequent removal of any residual uncapped RNA is a majorbottleneck for RNA production. Isolere Bio, Inc. specializes in designing innovative purification solutions thatcombine a protein affinity tag that is genetically fused to an elastin-like polypeptide (ELP). This reagent is capableof temperature or salt-controlled phase separation that enables capture of a specific target regardless of itscomplexity or the presence of other impurities. We will develop a novel combination of biopolymers, referred tohere as IVTagâ¢, that enables simultaneous RNA sequestration into concentrated, pure liquid droplets andmethylation of transcripts. Two ELP fusion proteins will be developed: one will contain an RNA-binding motif andthe second an O'-methyltransferase (O'-MTase) enzyme, responsible for 5'-capping of RNA. We will thendevelop platform methods for simultaneous purification of RNA into droplets and high efficiency capping of thesequestered RNA. This Phase I program is designed to demonstrate compatibility of this essential IVT enzymeand complexing it with Isolere's non-chromatographic purification methods. Key activities of the proposal includesynthesizing, purifying, and testing each biopolymer fusions' functionality and then evaluating their efficacy whenapplied in tandem and comparing process efficiency to the industry standard method. If successful, additionalfunctionality 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 totranscripts). 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 manufacturingworkflow 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 reagentshave proven to be easily manufacturable and re-usable for multiple purification cycles, reducing costs of goodssignificantly compared to affinity resins. The scalability, robust performance, and cost-effectiveness of theproposed product is an excellent fit for accelerating the development of therapeutic RNA candidates from benchscale discovery through large-scale manufacturing of commercial products.
Public Health Relevance Statement: Project Narrative
As RNA becomes a mainstream therapeutic, manufacturing methods must become cost-effective and scalable
to keep pace with an increasing number of patients, up to and including vaccination of the global population.
Isolere Bio, Inc. has developed several purification solutions that feature an affinity tag fused to a smart
biopolymer; this reagent specifically endows any target biologic with reversible soluble to insoluble behavior that
enables affinity capture, concentration, and contaminant removal to occur in micro-crucible droplets. This Phase
I research program will establish proof-of-concept of Isolere's scalable and efficient approach to manufacturing
RNA drugs and vaccines.
Project Terms: Antibodies ; Behavior ; Biopolymers ; Chromatography ; Cloning ; cost effectiveness ; DNA ; Deoxyribonucleic Acid ; Pharmaceutical Preparations ; Drugs ; Medication ; Pharmaceutic Preparations ; drug/agent ; Elastin ; Engineering ; Enzymes ; Enzyme Gene ; Escherichia coli ; E coli ; E. coli ; Goals ; In Vitro ; Ligands ; Mainstreaming ; Educational Mainstreaming ; achievement Mainstream Education ; Methods ; Methylation ; Methyltransferase ; EC 2.1.1 ; methylase ; transmethylase ; Multienzyme Complexes ; enzyme complex ; Optics ; optical ; Pain ; Painful ; Patients ; Poly A ; Poly(rA) ; polyadenylate ; Polynucleotide Adenylyltransferase ; ATP-RNA Adenylyltransferase ; Poly A Polymerase ; Polyadenylate Polymerase ; Polyadenylate Synthetase ; Riboadenylate Transferase ; Production ; Proteins ; Reagent ; Research ; Plant Resins ; resin ; RNA ; Non-Polyadenylated RNA ; RNA Gene Products ; Ribonucleic Acid ; Messenger RNA ; mRNA ; Sodium Chloride ; salt ; Mass Spectrum Analysis ; Mass Photometry/Spectrum Analysis ; Mass Spectrometry ; Mass Spectroscopy ; Mass Spectrum ; Mass Spectrum Analyses ; Tail ; Technology ; Temperature ; Testing ; Genetic Transcription ; Gene Transcription ; RNA Expression ; Transcription ; Translations ; Vaccination ; Vaccines ; Vision ; Sight ; visual function ; Measures ; Price ; pricing ; Chimeric Proteins ; Chimera Protein ; Fusion Protein ; base ; improved ; Left ; Residual state ; Residual ; Phase ; Biological ; Series ; Reporter Genes ; Ensure ; Poly(A) Tail ; Therapeutic ; fluid ; liquid ; Liquid substance ; Genetic ; programs ; Complex ; Reaction ; light scattering ; Performance ; success ; Speed ; novel ; Abscission ; Extirpation ; Removal ; Surgical Removal ; resection ; Excision ; Molecular Interaction ; Binding ; Pharmaceutical Agent ; Pharmaceuticals ; Pharmacological Substance ; Pharmacologic Substance ; polypeptide ; Polymerase ; Length ; Affinity ; Data ; Recombinants ; in vivo ; Small Business Innovation Research Grant ; SBIR ; Small Business Innovation Research ; Transcript ; Viral Vector ; Process ; cost ; design ; designing ; manufacturing process ; scale up ; cost effective ; Population ; innovation ; innovate ; innovative ; therapeutic development ; therapeutic agent development ; operation ; biophysical properties ; biophysical characteristics ; biophysical characterization ; biophysical measurement ; biophysical parameters ; Industry Standard ;
