Capillary Electrophoresis-Sodium Dodecyl Sulfate with Laser-Induced Fluorescence Detection as a Highly Sensitive and Quality Control-Friendly Method for Monitoring Adeno-Associated Virus Capsid Protein Purity.

Abstract

The capsid protein purity of adeno-associated virus (AAV) is considered a critical quality attribute of AAV-based gene therapy products. However, the analytical methods currently available to monitor the viral capsid proteins, which are present in extremely low concentrations, have limited sensitivity and robustness, thus limiting their general applicability. As a result, there is an urgent need to develop robust separation methods with highly sensitive detection. In this article, we describe the first denaturation and fluorescence labeling procedure for AAV capsid proteins using the pyrylium dye Chromeo™ P503, enabling the establishment of the first capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) method combined with laser-induced fluorescence (LIF) detection for AAV. Upon optimization using a quality-by-design approach, the newly developed method features a simple and robust one-step sample preparation workflow resulting in consistently labeled and denatured viral protein samples, which can subsequently be separated and quantified by CE-LIF. The method has been validated to be accurate and precise with a linear range of 50-150% of the nominal concentration of 2.0 × 1011 vector genomes per mL (vg/mL). The detection limit and quantitation limit were established to be 8.0 × 107 vg/mL (∼0.8 ng/mL) and 4.2 × 108 vg/mL (∼4 ng/mL), respectively, representing the highest sensitivity achieved for AAV capsid protein quantitation reported to date and a linear dynamic range of 8.0 × 107-3.0 × 1011 vg/mL. A comparison of the CE-SDS LIF method with existing methods, such as CE-SDS ultraviolet and sodium dodecyl sulfate-polyacrylamide gel electrophoresis with SYPRO Ruby stain, indicated that the new method has superior resolution and a significant increase in signal intensity. Capsid protein purity analysis of multiple AAV serotypes, including AAV5, scAAVrh10, AAV2, and AAV6, has been demonstrated for the first time using the same method, indicating the newly developed AAV labeling procedure and CE-LIF analysis could serve as a Quality Control-friendly platform and best-in-class analytical method for the control of AAV capsid protein purity.