Characterization of AAV vectors: A review of analytical techniques and critical quality attributes

Abstract

Standardized evaluation of adeno-associated virus (AAV) vector products for biotherapeutic application is essential to ensure the safety and efficacy of gene therapies. This includes analyzing 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, optimization of current assays, and development of reference materials. Highly precise methods are necessary for measuring the empty/partial/full capsid ratios and the titer 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 characterize AAV vectors by informing process development and facilitating the generation of reference materials for assay validation and calibration.

Keywords: AAV; CQAs; analytical methodologies; capsid identity; characterization; critical quality attributes; gene therapy; genetic identity; viral vector.

Graphical abstract

Figure 1

Schematic of the genome organization of wild-type AAV The genome of adeno-associated viruses (AAVs) consists of two main genes, rep and cap, flanked by T-shaped hairpin structures called inverted terminal repeats (ITRs). The rep gene encodes for four non-structural Rep proteins (Rep 78, Rep 68, Rep 52, and Rep 40), while the cap gene encodes three capsid proteins (VP1, VP2, and VP3). Alternative open reading frames (ORFs) of the cap gene encodes for the assembly-activating protein (AAP) and for the membrane-associated accessory protein (MAAP).

Figure 2

Schematic of the diverse AAV products that can be found in an AAV production batch Product-related impurities can include empty capsids, capsids containing partial genomes, and capsids encapsidating residual DNA from the rAAV production process.

Figure 3

Schematic representation of protein characterization workflows for rAAV preparations Capsid proteins are first identified using SDS-PAGE or western blotting. Serotype identification is accomplished via intact LC-MS analysis, whereas identification of PTMs, HCPs, and protein sequencing is achieved through liquid chromatography-tandem mass spectrometry (LC-MS/MS).