While recombinant adeno-associated virus (AAV) holds significant promise for effective and durable gene delivery for gene therapy, a thorough understanding of the critical quality attributes (CQAs) along with the degradation pathways of AAV under the various stresses that may occur during manufacturing, storage, and handling remains limited. To address this gap, we performed a comprehensive forced degradation study to elucidate the degradation pathways of AAV8 under a series of stress conditions, such as oxidation, extreme pH, high temperature, freeze-thaw, and agitation. Our results show that, under these stress conditions, distinct post-translational modifications (PTM), including methionine oxidation, asparagine deamidation, and aspartic acid isomerization, along with multiple physical degradation pathways, including capsid aggregation, viral protein fragmentation, and genome DNA leakage, could occur. Alterations in AAV8 biological activity were frequently attributed to the combination effect from chemical and physical degradation mechanisms. The results from this study provide a valuable insight into the establishment of stability-indicating methods and the identification of CQAs for AAV. It will also support the development of robust manufacturing process as well as stable and efficacious AAV gene therapy drug products.
Lai et al. (Thu,) studied this question.