Variable pathlength technology for in-/atline AAV characterization: Advancing process analytical technology in gene therapy manufacturing

Abstract

This project explored a variable pathlength UV absorbance-based Process Analytical Technology tool (SoloVPE®) for quantifying Vector Genome titer and estimating the percentage of full capsids in AAV samples, aiming to enhance Critical Quality Attributes monitoring in Adeno-Associated Virus production. Results from SoloVPE® were compared to an established orthogonal method, Size Exclusion Chromatography coupled to a Multi-Angle Light Scattering detector (SEC-MALS) to evaluate accuracy and reproducibility. Initial findings indicated that SoloVPE® results correlate well with SECMALS, maintaining a deviation within ±10%. However, performance was varied across sample types and storage conditions. For VG quantification, SoloVPE® exhibited excellent linearity (R² > 0.99) which was maintained over at least a one-log range of concentrations, validating its use in dynamic manufacturing environments. On the other hand, % full capsid estimations showed less reproducibility, also in comparison to the orthogonal Mass Photometry platform, and a tendency for underestimation compared to SEC-MALS results. The limitations of SoloVPE® stem from its inability to distinguish between full and partially filled capsids, alongside potential stability issues related to sample handling and storage. Despite these challenges, the SoloVPE® oƯers significant advantages in speed and ease of use, positioning it as a valuable PAT tool for in-process monitoring and screening in AAV production.