Development of an icIEF assay for monitoring AAV capsid proteins and application to gene therapy products

Adeno-associated virus (AAV) gene therapy vectors, which contain a DNA transgene packaged into a protein capsid, have shown tremendous therapeutic potential in recent years. Methods traditionally used in quality control labs, such as high-performance liquid chromatography (HPLC) and capil-lary electrophoresis (CE), do not provide a complete under-standing of capsid viral protein (VP) charge heterogeneity. In the present study, we developed simple, one-step sample prep-aration and charge-based VP separation using imaged capillary isoelectric focusing (icIEF) for monitoring AAV products. The robustness of the method was confirmed through a design of experiments (DoE) exercise. An orthogonal reverse-phase (RP) HPLC method coupled with mass spectrometry was devel-oped to separate and identify charge species. Additionally, capsid point mutants demonstrate the capability of the method to resolve deamidation at a single site on the viral proteins. Finally, case studies using two different AAV serotype vectors establish the icIEF method as stability indicating and demon-strate that increases in acidic species measured by icIEF corre-late with increased deamidation, which, we show, results in decreased transduction efficiency. The addition of a rapid and robust icIEF method to the AAV capsid analytical toolkit enables development and consistent manufacturing of well -characterized gene therapy products.