Characterisation of AAV Vectors: A Review of Analytical Techniques and Critical Quality Attributes

Abstract

Standardised evaluation of adeno-associated virus (AAV) vector products for biotherapeutic application is essential to ensure the safety and efficacy of gene therapies. This includes analysing the critical quality attributes of the product. However, many of the current analytical techniques used to assess these attributes have limitations, including low-throughput, large sample requirements, poorly understood measurement variability, and lack of comparability between methods. To address these challenges, it is essential to establish higher-order reference methods that can be used for comparability measurements, optimisation of current assays, and development of reference materials. Highly precise methods are necessary for measuring the empty/partial/full capsid ratios and the titre of AAV vectors. Additionally, it is important to develop methods for the measurement of less established critical quality attributes, including post-translational modifications, capsid stoichiometry, and methylation profiles. By doing so, we can gain a better understanding of the influence of these attributes on the quality of the product. Moreover, quantification of impurities, such as host cell proteins and DNA contaminants, is crucial for obtaining regulatory approval. The development and application of refined methodologies will be essential to thoroughly characterise AAV vectors, by informing process development and facilitating the generation of reference materials for assay validation and calibration.