Forced degradation analysis of recombinant adeno-associated virus serotype 8 based on analytical anion exchange chromatography coupled to orthogonal characterization

As a leading vector for gene therapy, recombinant adeno-associated virus (rAAV) has been widely investigated and used. Forced degradation analysis is required to understand the molecular properties and stability of rAAV. In this study, we have applied an analytical anion exchange chromatography (AEX) method to evaluate the degradation behavior of rAAV serotype 8 (rAAV8) stored at a relatively high temperature (40 °C), coupled to orthogonal characterization. This versatile methodology was then used to assess the full-particle (FP) property of rAAV8 after accelerated storage under a wide range of pH conditions (pH 2.5-9.5). Under neutral and basic conditions, prolongation of chromatographic retention and decreases in peak area were identified, and were associated with changes in deamidation-related surface charge, aggregation, and nonspecific adsorption, which is likely caused by the externalization of viral proteins 1 and 2 (VP1/VP2). In contrast, unusual chromatographic variations under acidic conditions were identified, and suggested substantial degradation, including fragmentation and the release of encapsidated DNA, as well as the cleavage of VP1 and VP2. Thus, we have demonstrated the potential of the combined methodology and provided valuable insight into rAAV stability during manufacturing and storage.