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 capillary electrophoresis (CE), do not provide a complete understanding of capsid viral protein (VP) charge heterogeneity. In the present study, we developed simple, one-step sample preparation 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 developed 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 demonstrate that increases in acidic species measured by icIEF correlate 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.

Silicon Photonic Microring Resonator Arrays as a Universal Detector for Capillary Electrophoresis.

Electrophoretic separations conventionally rely on chromogenic, fluorogenic, or redox properties for analyte detection that, in many instances, involve chemical modification of samples prior to analysis. For analytes natively lacking chemical signatures, refractive index-based measurements are appealing as a method to detect these molecules without pretreatment. Microring resonators are a type of whispering gallery mode sensor capable of detecting bulk changes in refractive index. Here, we demonstrate the use of silicon photonic microring resonator arrays as a postcolumn detector for capillary electrophoresis. In this approach, we establish the universal detection capabilities of microrings through calibration with analytes lacking unique spectral signatures. Separations of small molecule mixtures are demonstrated using capillary zone electrophoresis. For these separations, the microring resonators maintain a linear response over several orders of magnitude in concentration for three candidate small molecules. Successful separation of three sugars with direct detection is also demonstrated. We further present the successful separation and detection of three model proteins, exemplifying the promise of microring resonators arrays as a biocompatible detector for capillary electrophoresis. Additionally, the spatially offset, array-based nature of the sensing platform enables real-time analysis of analyte mobility and performance characterization-a combination that is not typically provided using single-point detectors.