Gene therapies remain promising candidates for a broad range of intractable diseases, however their complexityrenders traditional biopharmaceutical manufacturing procedures inefficient, costly, and impractical. Given that asingle dose of an AAV-delivered gene therapy may require purification of virus from 60 L of culture media ormore, 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 AAVpurification, there is currently no "gold standard" method in commercial production. These limitations of currentmethods have motivated Isolere Bio, Inc. to develop an innovative purification platform for viruses: ViraTagâ¢.ViraTag⢠involves two sequential steps. First, the viruses are affinity captured by a custom protein reagent andthe user triggers a liquid-liquid phase transition by a simple environmental change, such as the addition of saltor heat. The virus particles are sequestered into water-immiscible droplets, with host cell proteins and othercellular 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 toaccommodate 103 to 105 L of input media; 2) mild elution conditions to preserve vector function and therapeuticefficacy; 3) validated compatibility with automated downstream purification instrumentation and workflows usedin biomanufacturing, and 4) the potential to isolate capsids containing therapeutic nucleic acid payloads awayfrom empty capsids. During Phase I of this SBIR Fast track proposal, we will engineer a new ViraTag⢠reagentcapable of affinity capture and phase separation of AAV particles, and evaluate its efficiency in AAV purificationfrom 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 thatcannot be scaled up. Phase II will focus on scale-up of ViraTag⢠manufacturing, by developing workflows andproduct form factors for various application scales, and benchmarking the purity of final products against currentmethods. Phase II will also include external validation of the process by laboratories representative of theproduct's target customers, ranging from bench-scale academic research to large-scale biopharma.
Public Health Relevance Statement: 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 ViraTagâ¢, 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.
Project Terms: Dependovirus ; Adeno-Associated Viruses ; Dependoparvovirus ; adeno associated virus group ; Beds ; Behavior ; Biological Products ; Biologic Products ; Biological Agent ; biopharmaceutical ; biotherapeutic agent ; Biotechnology ; Biotech ; Buffers ; Capsid ; Cell Culture Techniques ; cell culture ; Cells ; Cell Body ; Centrifugation ; Centrifugation Fractionation ; Chromatography ; Affinity Chromatography ; affinity purification ; Culture Media ; growth media ; Disease ; Disorder ; DNA ; Deoxyribonucleic Acid ; Elastin ; Engineering ; Environment ; Feedback ; Fermentation ; Filtration ; Filtration Fractionation ; gene therapy ; DNA Therapy ; Gene Transfer Clinical ; Genetic Intervention ; gene-based therapy ; genetic therapy ; genomic therapy ; Gold ; Industry ; instrumentation ; Laboratories ; Methods ; Methodology ; Peptides ; Production ; Proteins ; Reagent ; Recombinant Fusion Proteins ; Research ; Research Personnel ; Investigators ; Researchers ; Safety ; Serotyping ; Sodium Chloride ; salt ; Technology ; Ultracentrifugation ; Universities ; Virion ; Virus Particle ; Virus ; Water ; Hydrogen Oxide ; Price ; pricing ; Custom ; Procedures ; virus culture ; Left ; Clinical ; Phase ; Training ; Stimulus ; fluid ; liquid ; Liquid substance ; Contracting Opportunities ; Contracts ; Scientist ; Protocol ; Protocols documentation ; Viral ; Best Practice Analysis ; Benchmarking ; particle ; Performance ; success ; aqueous ; novel ; bioprocess ; flasks ; Documentation ; Manufacturer ; Manufacturer Name ; Coat Proteins ; Viral Coat Proteins ; Viral Outer Coat Protein ; Capsid Proteins ; Molecular Interaction ; Binding ; Pharmaceutical Agent ; Pharmaceuticals ; Pharmacological Substance ; Pharmacologic Substance ; Phase Transition ; Address ; Dose ; Affinity ; Harvest ; Collection ; Gene Transduction Agent ; Gene Therapy Vectors ; Gene Transduction Vectors ; Phage Display ; Small Business Innovation Research Grant ; SBIR ; Small Business Innovation Research ; Validation ; Viral Vector ; Process ; Development ; developmental ; environmental change ; vector ; cost ; design ; designing ; Treatment Efficacy ; intervention efficacy ; therapeutic efficacy ; therapy efficacy ; scale up ; cost effective ; innovation ; innovate ; innovative ; Biomanufacturing ; therapeutic target ; product development ; preservation ; nucleic acid-based therapeutics ; therapeutic nucleic acids ;
