The Characterization of AAV Capsid Individual VP-Specific Charge Heterogeneity Using a High-Throughput Reduced Denatured iCIEF–Western Method

In recent years, advancements in gene therapy have highlighted the important role of adeno-associated viruses (AAVs) due to their favorable characteristics, such as low immunogenicity compared to other viral vectors, e.g., lentivirus and HSV, and the ability to maintain gene expression in a variety of tissues. However, the production of recombinant AAVs in biological systems can lead to variability in the biophysical properties of viral capsid proteins, primarily due to post-translational modifications (PTMs) and cleavage events during downstream processing and storage. A critical quality attribute of AAV capsids is charge variant heterogeneity, which is significantly influenced by PTMs like deamidation, phosphorylation, acetylation and glycosylation. These modifications can impact the safety and efficacy of the viral vectors. The traditional imaged capillary isoelectric focusing (iCIEF) method, which uses absorbance or fluorescence detection, has been the primary choice for characterizing charge variants. However, it often lacks the sensitivity and resolution needed for AAVs’ charge variants. We introduce an optimized, highly sensitive capillary-based Western method to measure the apparent isoelectric point (pI) and detect charge heterogeneity at the individual VP protein level under reduced denatured conditions. This approach involves generating and purifying polyclonal antibodies to detect charge variants specific to the VP1, VP2, and VP3 proteins across different AAV serotypes, including AAV1, AAV8, and AAV5. This method is a valuable tool for the characterization of AAV capsids and can be utilized for the stability assessment and analysis of in-process and purified samples during process optimization from a charge heterogeneity perspective.