Comprehensive stress study on recombinant adeno-associated virus vectors: Evaluating the capabilities of established analytical techniques

Recombinant adeno-associated virus (rAAV) vectors are prominent vectors for in-vivo gene therapies. However, achieving consistent product quality is a major challenge due to the inherent complexity of their structure and the associated production process. During manufacturing and storage, virus particles are exposed to a variety of stresses, which can have a direct impact on product attributes, thereby potentially also affecting safety and efficacy. Current understanding of stress-induced degradation for rAAVs is still fragmented, both on a mechanistic level and regarding the identification of the most appropriate analytical tools to detect the underlying changes. To address these gaps, the impact of different stress conditions, namely freeze-thaw, elevated temperature, high/low pH and light exposure, was tested on two different serotypes. For this, a comprehensive panel of orthogonal analytical methods was applied to identify significant changes in capsid and genome titer, full-to-total ratio, content of aggregates, payload integrity and post-translational modifications. By correlating this extensive dataset with potency results, this study provides detailed insights into the degradation behavior of the employed rAAVs and the suitability of state-of-the-art analytical techniques to indi-
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cate the stability of the product. Overall, substantial differences in stress responses were observed between the two serotypes investigated, and changes in transduction efficiency were mostly represented inadequately at the structural or molecular level by the analytical methods employed. Based on these findings, this
study serves as a framework for refining the analytical control strategy of rAAV-based gene therapies and for developing robust, stability-indicating assays.