Systematic formulation optimisation to enhance buffer exchange recovery and storage stability for adeno-associated...

Recombinant adeno-associated virus (AAV) vectors have emerged as the leading platform for gene therapy applications due to their non-pathogenic nature and efficacy across a variety of tissues. To date, a growing body of AAV-based therapies have been approved by regulatory authorities. However, therapeutic AAV administration currently requires high vector doses with drug product concentrations in vials often exceeding 1 x 10¹³ viral genomes/mL. Such high product concentrations can induce aggregation and in turn contribute to undesirable immune responses. Drug product formulation plays a critical role in maintaining AAV vector stability, functionality, and shelf-life. However, the number of excipients tested in AAV formulation development remains limited, and their concentrations are often assessed within a very narrow concentration range. In this study, we aimed to expand the formulation toolbox for AAV2 and investigated up to twenty excipients across six distinct categories for enhanced formulation compositions. Here we outline a systematic framework for screening, refining and optimising excipient concentrations through a sequential design of experiments (DOE) methodology of three steps of experimentation. Excipient effects and interactions were evaluated based on AAV thermal stability as measured by differential scanning fluorimetry (DSF), and AAV ultrafiltration/diafiltration (UF/DF) process recovery via size exclusion-high-performance liquid chromatography (SEC-HPLC). In this manner, an optimised formulation was developed with a melting temperature (T_m) exceeding 70 °C and recovery > 90 % post ultrafiltration/diafiltration. Accelerated degradation studies at 40 °C demonstrated the excellent stability of OptiDOE, the optimised formulation developed in this study, with AAV retaining at least 60 % infectivity and over 87 % viral particle concentration (>1 x 10¹³ vp/mL) after 14 days. Our findings demonstrate a methodical approach that can be adapted to explore a large array of excipients and concentrations to improve the manufacturability and shelf-life of future AAV products.