Rapid At‐Line AAVX Affinity HPLC: Enabling Process Analytical Technology for Bioprocess Development of Adeno‐Associated Virus Vectors

Recombinant adeno-associated virus (rAAV) vectors have emerged as a new class of therapeutic modal with the promise to treat or even cure hereditary and acquired diseases, but their consistent and efficient production remains challenging. To address these inadequacies, the implementation of process analytical technology (PAT) principles for the development of rAAV-based gene therapies holds the prospect of promoting greater product and process understanding. However, a substantial lack of suitable analytical tools during both upstream and downstream processing (DSP) hinders the ability to fully realize the potential of PAT for rAAVs. To fill this gap, our recently described AAVX affinity-based high-performance liquid chromatography (HPLC) method was assessed as an at-line PAT tool to determine the capsid titer and the percentage of filled capsids at various stages of the production process. Leveraging the fast and robust provision of these parameters, even for challenging samples, the benefits of this approach for improved process monitoring and control were demonstrated for samples generated both during fermentation and DSP. Given the versatility of our developed analytical method for different rAAV serotype and payload combinations, we eventually highlight its expansive opportunities to streamline process development and therefore contributing to high-quality and cost-efficient production of rAAV-based gene therapies.