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.

AAVs have emerged as a promising platform for in vivo gene delivery. Currently, a total of ten AAV gene therapy products have been approved by regulatory authorities [1][2]. As demand for AAV-delivered therapeutics grows, ongoing optimization of AAV manufacturing is essential. The manufacturing of AAV DP is a multi-faceted process encompassing several critical steps including plasmid manufacturing, upstream processing (e.g., cell expansion, transfection, and viral vector production), downstream processing (e.g., cell lysis, filtration, and purification), formulation, and fill/finish [3][4][5][6][7]. Prior to formulation or compounding to generate DS, the AAV UF/DF pool can be refrigerated for short-term or frozen at ≤ -60°C for extended periods. After completion of formulation, the DS is stored long-term at ≤ -60°C. To produce AAV DP, the DS is thawed and then undergoes pooling, mixing, and sterile filtration prior to DP filling stage [8]. Unlike the well-established manufacturing processes for mAbs, the manufacturing of AAV DS is evolving due to ongoing manufacturing challenges especially with yield, scale-up capability, and manufacturing consistency [3][4]. One of the challenges in AAV DS manufacturing is selecting an appropriate storage container for AAV DS to ensure compatibility and stability during manufacturing process and long-term storage at deep-frozen temperatures (≤ -60°C). Although the storage containers for mAb DS have been extensively studied, with common viable options including plastic containers such as polycarbonate bottles and bags with EVA product contact layer [9][10][11][12][13][14][15], there is limited published information regarding the optimal storage container solutions for AAV DS.

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