Identification of relevant analytical methods for adeno-associated virus stability assessment during formulation development

ABSTRACT Formulation development for gene therapy viral vectors requires high-performance analytical methods that indicate or predict virus stability. Additionally, if testing involves multiple excipients, sample throughput must be sufficiently high, and the method should require low amounts of virus material. These are challenging and sometimes conflicting requirements, especially in the analysis of adeno-associated viral vectors, which are currently the most prominent viral vectors in gene therapy development. In this exploratory study, we evaluated the ability of different analytical methods to indicate or predict the stability of the adeno-associated viral vectors and identified highly suitable techniques that fulfilled the requirements for analysis during formulation development. As functionality assays, such as reporter gene assays, are already established for rapid quality control assessments, they are not addressed in this study. From this study, we conclude that two methods have great potential to predict the stability of adeno-associated viral vectors: extrinsic differential scanning fluorimetry using SYBR Gold as a DNA binding fluorescent dye to detect genome release and nano differential scanning fluorimetry to investigate capsid unfolding behavior (based on intrinsic protein fluorescence). Size-exclusion chromatography, using multi-angle light scattering and UV detection, was found to be stability-indicating for particle size distribution and determining genome load of adeno-associated viral vectors.IMPORTANCEViral vectors are of growing importance in the areas of gene therapy, oncology, and vaccine development. However, these vectors are very unstable and usually have to be stored frozen at very low temperatures due to sub-optimal formulation conditions, in many cases. The development of superior formulations for viral vectors requires high-performance analytical methods. In this study, we evaluated relevant analytical methods with respect to sample throughput, material consumption, and applicability for viral vector formulation development. To our knowledge, this is the first time that methods for viral vector analysis were categorized according to their power to predict or indicate time-dependent long-term stability. This categorization of analytical methods is essential to rationalize, accelerate, and enhance the formulation development of viral vectors. Therefore, the studies in this article are prerequisites for the development of more stable viral vectors for gene therapy and vaccines and higher yields in manufacturing.