Development of a Viral Vector Sizing Tool to Improve Gene Therapy Analytics and Reduce the Cost of Drug Development

ABSTRACT NanoEngineering is commercializing nanoparticle characterization technology for gene therapy vector analytics. Licensed from Yale, our proprietary electrospray differential mobility analysis (ES-DMA) method quickly and easily measures the size of virus capsids at angstrom-scale resolution. This SBIR proposal builds on our preliminary success toward developing our portable benchtop instrument, NanoRanger, as a standard tool to monitor and improve the purity of viral vectors and reduce production costs. Adeno-Associated Virus (AAV) is the most commonly used viral vector for in vivo gene therapy but commercial production methods are currently unable to achieve complete separation of “full” AAV capsids from contaminating “empty”, and “partial” / “over- filled” particles. These species are therefore often present in clinical preparations, reducing efficacy, increasing production costs, and posing serious health risks for patients. For effective analysis by ES-DMA it was necessary that upon electrospray ionization, AAV vectors would exhibit measurable differences in size based on their DNA cargo. Research carried out with our partners at MegaDalton Solutions and Vector Biolabs on 11 cargo- containing AAV9 samples and certified empty reference materials has now shown that spectra obtained from full AAVs are readily distinguishable from the empty capsid profile and display shifts in particle diameter strongly correlated with the genomic length of packaged DNA cargo. Direct comparison of full / empty capsid ratios was assessed against parallel testing by charge detection mass spectrometry (CD-MS), a known gold-standard. Strikingly, these results closely matched those obtained by ES-DMA for 8 of 11 samples. Successful commercial development of this technology will help the industry meet cost reduction goals by shortening feedback about process improvements in downstream purification. Long term, NanoRanger will target upstream production monitoring where metrology tools are needed most. The specific aims of this project are first to establish a reproducible protocol for AAV analysis across various serotypes and genomic cargos, and second to confirm robust mass/size correlation through continued parallel CD-MS testing, targeting a 95% Confidence Interval for mass/size correlation and submission for peer-reviewed publication. Third, we will deploy a NanoRanger prototype to Vector Biolabs for on-site beta testing at their AAV production facility in Malvern, PA. We expect Phase 1 to establish ES-DMA as a novel QC method for AAV analytics and to confirm the unique advantages of our approach for bio-nanoparticle characterization, ultimately helping to improve safety and reduce costs of lifesaving therapeutics for millions of patients worldwide. We anticipate a total annual sales opportunity of $50M based on a target market of over 500 analytical labs and 1000’s of bioreactors. Phase 2 will further develop and market NanoRanger as a new gold standard for AAV analytics. We will also continue process development for other applications in AAV research and shelf-life stability QC, develop autonomous operation for real-time production monitoring, and expand our focus to other viral and non-viral gene therapy vectors.