Integrating High-Throughput Chromatography and Data Science in a Platform for the Rapid and Accurate Quantification of Full, Partial, and Empty AAV Capsids

Propose a platform to streamline the design of chromatographic methods for quantifying gene-loaded (full), partially loaded (partial), and empty Adeno-Associated Virus (AAV) capsids.

Industry Need

The success of AAV-based gene therapies relies on bioprocess technologies that can produce high-purity, cell-transducing capsids at clinically relevant scales. However, current expression systems generate a mix of full, partial, and empty capsids, whose proportions vary with serotype, transgene, and process conditions, posing safety risks such as immunogenicity and genotoxicity. Removing these impurities requires a complex anion-exchange chromatography (AEX) enrichment step, which must be tailored to specific AAV designs and sample characteristics. Developing effective AEX protocols is challenging due to numerous interdependent parameters, including stationary phase properties, flow rates, buffer compositions, and elution formats.

Approach

To address the challenges in AAV capsid purification, this project will leverage the combined expertise and resources of NIIMBL academic and industry partners, including advanced chromatographic technologies, modeling tools, and a diverse library of model AAVs. We will establish the Platform for Analytical Chromatographic Testing (PACT) to rapidly screen and validate AEX protocols across varied AAV designs, generating a comprehensive dataset of in-line, at-line, and off-line analytics. This data will inform the development of the Rulebook of AAV Analytical Chromatography (RAAC), a predictive framework linking chromatographic parameters to capsid purity outcomes. Together, PACT and RAAC will transform AEX method development from empirical trial-and-error to a systematic, data-driven approach, accelerating gene therapy manufacturing and supporting NIIMBL’s broader data initiatives.

Impacts

Accelerate Drug Development by transforming the development of chromatography methods for the isolation/quantification of full capsids from an empirical approach to a systematic, data-driven, and product-oriented method.

Advance Analytics for Process Optimization by providing a rapid and accurate At-Line assay, adaptable across serotypes and transgenes, that quantifies full, partial, and empty AAV capsids with agreements of > 95% with mass photometry and > 90% with analytical ultracentrifugation.

Enhance Process Productivity and Robustness, and Ensure Product Quality by leveraging functional correlations linking AAV design and capsid properties, analytical chromatograms and scattergrams, CQAs measured by state-of-the-art analytics, and performance of AAV purification processes.