Development of a Rapid Adeno-Associated Virus (AAV) Identity Testing Platform through Comprehensive Mass Analysis of Full-length AAV Capsid Proteins

Adeno-associated viruses (AAVs) are commonly used as vectors for the delivery of gene therapy targets. Characterization of AAV capsid proteins (VPs) and their post-translational modifications (PTMs) has become a critical attribute monitored to evaluate product quality. To accommodate the growing use of AAV delivery systems, rapid analytical methods that comply with good manufacturing practice (GMP) standards are needed. As shown in previous studies, mass protein liquid chromatography-mass spectrometry (LC-MS) analysis of full-length AAV VPs provides both quick and reliable serotype identification as well as proteoform information of each VP. However, for AAV2, separation of VP2 and VP1 has proven difficult to obtain, with successful separation only achieved when using mobile phase modifiers incompatible with MS. This incompatibility hinders in-depth AAV VP characterization by suppressing ion signal, thus reducing VP proteoform identifications through the generation of low intensity MS spectra. Using AAV2 as a test case, we demonstrate how coupling hydrophilic interaction liquid chromatography (HILIC) with MS compatible difluoroacetic acid (DFA) as a mobile phase modifier, achieves complete separation of the three AAV VPs while also generating high quality MS spectra. Employing increased MS resolving power enabled improved identification of VP proteoforms whose PTMs were confirmed using peptide mapping. The LC-MS workflow was further transformed to develop an assay using GMP compliant software capable of rapid AAV serotype profiling. Incorporated into this method is the ability to perform serotype PTM characterization if desired. Such a platform provides product quality control capabilities that are easily accessible within a regulatory setting.