Poster
13

Characterization of Adeno-Associated Virus (AAV) Using Capillary Electrophoresis

Authors

( Melovic1
1 Ramcon, Sweden

Abstract

INTRODUCTION

Fast growth in cell and gene therapy industry has generated an urgent need for fast and robust analytics for characterization of both vectors and various types of nucleic acid biotherapeutics such as oligonucleotides and plasmid DNA. Adeno-associated virus (AAV) is one of the most widely used gene delivery vehicles for gene therapy because of its non-pathogenicity, low immunogenicity and different tropism to multiple cell types. Although there are efforts to develop methods for characterization of AAV, there are still limitations and drawbacks to some of these analysis. In addition, the different workflows are done on different platforms. Purity analysis of the AAV viral proteins is important for quality assurance and safety of AAV products. Although SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) has been used for AAV capsid protein analysis in the industry, CE-SDS (Capillary Electrophoresis - Sodium Dodecyl Sulfate) method on the SCIEX PA800 Plus provides automated separation of proteins in the range of 10kD to 225kD with higher resolution, quantitation capability, better reproducibility and is less labor intensive than traditional SDS-PAGE. The CE-SDS method using UV or PDA detector and stacking injection technology could provide good results for AAV sample with titer greater than 1X1012 GC/mL. However, for in-process AAV product analysis, higher sensitivity is required for purity analysis of AAV with concentration as low as 1X1010 GC/mL. Here we present CE based workflows for characterization of AAV viral proteins using a UV detector, and alternatively utilizing a dye for sample labeling with LIF detector when an increase in sensitivity is required. These methods provide a straight forward and easy sample preparation, excellent resolving power, good repeatability and linearity of absorbance response to sample concentration.

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