Comparative assessment of drug substance frozen storage containers for AAV8 manufacturing

Recombinant AAV has attracted significant interest, especially in recent years, due to their clinical success as the optimal gene therapy delivery system. This success has led to a pressing need to accelerate AAV production. Unlike monoclonal antibodies (mAbs), AAVs are protein – DNA complexes that have distinct stability features, and their bioprocesses are not yet fully understood. During AAV manufacturing, the ultrafiltration and diafiltration (UF/DF) pool and drug substance (DS) may be stored under refrigerated or frozen conditions for extended time. Additionally, they may also be subjected to manufacturing stresses such as agitation, extended room temperature hold, or freeze – thaw cycles prior to drug product (DP) manufacturing. Choosing suitable storage containers for AAV DS is crucial to ensure compatibility and stability of AAV DS when stored at deep-frozen temperatures (≤ -60°C) and under stress conditions. This study evaluated the performance of AAV8 DS in two common single-use containers for frozen storage, polyethylene (PE) and ethylene-vinyl acetate (EVA) bags, after extended storage at -80°C, 5°C and/or 25°C, multiple freeze – thaw cycles, or agitation to mimic the conditions that AAV8 DS may be exposed to during manufacturing processes. Results from this study revealed acidic pH drifts in AAV8 stored in the EVA bag due to acetate leachable, whereas pH remained stable when stored in the PE bag. A mitigation strategy was developed by increasing buffer capacity for AAV8 formulations, which successfully stabilized the pH drifts associated with the EVA bag.