SBIR/STTR Award attributes
PROJECT SUMMARY Gene therapies remain promising candidates for a broad range of intractable diseases, however their complexity renders traditional biopharmaceutical manufacturing procedures inefficient, costly, and impractical. Given that a single dose of an AAV-delivered gene therapy may require purification of virus from 60 L of culture media or more, there is an urgent and unmet need to streamline the process with a scalable, high throughput, and cost- effective solution. While ultracentrifugation and chromatography are the most common methods for AAV purification, there is currently no “gold standard” method in commercial production. These limitations of current methods have motivated Isolere Bio, Inc. to develop an innovative purification platform for viruses: ViraTag™. ViraTag™ involves two sequential steps. First, the viruses are affinity captured by a custom protein reagent and the user triggers a liquid-liquid phase transition by a simple environmental change, such as the addition of salt or heat. The virus particles are sequestered into water-immiscible droplets, with host cell proteins and other cellular contaminants left behind in the aqueous phase. Second, once contaminants have been washed away, the AAV are extracted from the droplets by lowering pH, which releases them from the capture reagent. ViraTag™ addresses key requirements for an ideal purification process, including: 1) linear scalability to accommodate 103 to 105 L of input media; 2) mild elution conditions to preserve vector function and therapeutic efficacy; 3) validated compatibility with automated downstream purification instrumentation and workflows used in biomanufacturing, and 4) the potential to isolate capsids containing therapeutic nucleic acid payloads away from empty capsids. During Phase I of this SBIR Fast track proposal, we will engineer a new ViraTag™ reagent capable of affinity capture and phase separation of AAV particles, and evaluate its efficiency in AAV purification from cell culture media. We will also evaluate the ability of this new affinity capture reagent to distinguish “full” versus “empty” capsids, an urgently needed capability currently only enabled by ultracentrifugation methods that cannot be scaled up. Phase II will focus on scale-up of ViraTag™ manufacturing, by developing workflows and product form factors for various application scales, and benchmarking the purity of final products against current methods. Phase II will also include external validation of the process by laboratories representative of the product’s target customers, ranging from bench-scale academic research to large-scale biopharma.PROJECT NARRATIVE Biomanufacturing of engineered viruses is a challenging and rate-limiting process which impacts the cost, safety and efficacy of gene therapies. Traditional ultracentrifugation and chromatography methods are currently employed for purification but are inefficient, costly, and impractical to scale up for gene therapy viral vectors. Isolere Bio, Inc. has developed ViraTag™, a novel non-chromatographic purification process capable of affinity- capture of adeno-associated viruses (AAV) and their subsequent isolation away from contaminants via conditional liquid-liquid phase transition. AAV capsids can be sequestered into ViraTag™ droplets away from contaminants and then subsequently released from ViraTag™ droplets in a user-triggered manner for collection of pure product. This technology addresses the currently unmet need for a cost-effective, high yielding, and scalable purification method for manufacturing gene therapy vectors.
