Enabling icIEF Peak Identification of AAV Capsid Proteins by Fractionation on MauriceFlex and Subsequent Analysis by LC–MS

A significant limitation of imaged capillary electric focusing (icIEF) is the inability to identify and characterize specific species in the electropherogram. This has led to the development of complementary ion-exchange chromatography (IEX)-based methods that are amenable to either fraction collection and subsequent characterization or online IEX coupled to mass spectrometry. To overcome this limitation while maintaining the use of icIEF, novel approaches, including an icIEF separation and fractionation technology (MauriceFlex, ProteinSimple), have been developed. This approach enables the fractionation of various icIEF peaks, which can then be characterized by mass spectrometry to confirm the identity of the separated charged species. Herein, the MauriceFlex technology was applied to adeno-associated viral (AAV) gene therapy products, which contain a DNA transgene packaged into a protein capsid and have shown tremendous therapeutic potential in recent years. Utilizing the MauriceFlex system, we developed an approach for the separation of charged species from AAV capsid viral proteins (VP) by icIEF and subsequent characterization by liquid chromatography and mass spectrometry (LC–MS). When applying the same sample preparation, charge profiles of AAV capsid proteins on the MauriceFlex instrument were demonstrated to be consistent with those from the original Maurice platform, the industrial gold standard. Optimization of the VP icIEF fractionation method required the development of a method for low concentration samples, optimization of mobilization conditions, enhancement of fraction recovery, and maintenance of protein stability post fractionation. Herein, we were able to successfully collect charge-separated VP fraction samples and subsequently analyze them by MS analysis. In addition, a workflow for AAV capsid protein characterization based on icIEF separation and fractionation coupled with downstream LC–MS has been established for the confirmation of VP identity and additional characterization of capsid protein heterogeneity.