10 Buyers’ Guide 2021
Ultrahigh Sensitivity Analysis of Adeno-associated Virus (AAV) Capsid Proteins by Sodium Dodecyl Sulphate Capillary Gel Electrophoresis
Tingting Li*, Marcia Santos and Andras Guttman SCIEX, Brea, CA 92821, USA *Corresponding author:
tingting.li@
sciex.com
Adeno-associated virus (AAV) is currently one of the most widely used delivery vehicles for gene therapy, featuring long-term expression of the transgene and excellent disease correction history. During vector production, several quality control (QC) parameters should be closely monitored to comply with clinical safety and efficacy requirements. Among them, purity analysis of the AAV viral proteins is important for quality assurance and safety of the products. This paper introduces two sodium dodecyl sulphate capillary gel electrophoresis (SDS-CGE) workflows for adeno- associated virus capsid protein analysis. One is utilising UV detection for the intact forms and the other one with fluorophore tagging for enhanced sensitivity laser induced fluorescence detection.
1. Introduction
AAVs are members of the parvovirus family, being among the smallest DNA viruses with a diameter of approximately 20 nm. Importantly, they are non- enveloped, replication-defective viruses. AAVs are composed of three capsid proteins surrounding the DNA genome of approximately 4.8 kilobases (kb) [1]. The three viral capsid proteins are 87kD (VP1), 73kD (VP2) and 61kD (VP3) in size and assembled in an approximately 1 : 1 : 10 molar ratio, respectively [2, 3]. Sixty of these subunits form a perfect icosahedral structure [4, 5]. During AAV manufacturing and release, impurities should be subject to rapid and high resolution analysis, including residual host cell and helper virus related proteins from the culture medium and purification process [6].
Various serotype AAVs have become attractive therapeutic vehicles for gene therapy development due to features such as long-term transgene expression and excellent disease treatment capability [7]. Pseudo-serotyping of adeno-associated viruses showed novel tropism and biology on individual genome/capsid configurations,
therefore, greatly improving the applicability and versatility of the system. Currently, thirteen human AAV serotypes have been identified increasing the applicability of this particular vector in gene therapy due to the distinct tropism of the different serotypes for various organs and tissues [8]. Cellular attachment of most AAV serotypes utilises binding to the carbohydrate moieties of various cell surface glycoproteins, an important first step towards successful transduction. AAV serotypes can be grouped by considering their receptor recognition as heparan sulphate proteoglycan (AAV2, AAV3,
Figure 1. SDS-CGE-UV separation of the AAV8 (trace A, 1 x1013 (trace B, 0.5 x1013
GC/mL) and AAV2 GC/mL) capsid protein samples. Conditions: SDS-MW gel buffer
system, 20 cm effective capillary length (30 cm total), 50 µm ID; UV detection at 214 nm; Separation voltage: 15 kV, temperature: 25ºC. Injection: water pre-injection for 0.4 min at 20 psi followed by sample injection for 1 min at 5 kV. Peaks: VP1, VP2 and VP3: virus capsid proteins, VP3’: virus capsid protein 3 with altered PTM.
AAV6, and AAV13) as well as for N-linked oligosaccharides: terminal sialic acid (AAV1, AAV4, AAV5, and AAV6) and terminal galactose (AAV9) types [9]. Therefore, qualitative and quantitative analysis of the virus capsid proteins and their distribution ratio is of high importance.
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