title: Adeno-associated virus capsid serotype identification: Analytical methods development and application.
abstract: Mass spectrometry (MS) has been utilized to address the need for a rapid and reliable assay to confirm the capsid serotype identity of recombinant AAV gene transfer vectors. The differences in the primary amino acid sequence of AAV serotypes generate a unique set of fragments with different masses upon proteolytic digestion, and by comparing the fragment masses against common and custom databases, reliable capsid serotype identification is achieved. Highly homologous serotypes, such as AAV1, AAV2, and AAV8, can be distinguished from each other, as well as from less homologous serotypes such as AAV4, and AAV5. Furthermore, analysis of the MS data for wild-type AAV4 compared to an AAV4 capsid with a single amino acid mutation demonstrates the sensitivity of the method and validates the relevance of the method in the context of retinal gene transfer. With an expanding repertoire of AAV serotypes, physicochemical methods for capsid analysis, such as MS, are highly desirable and do not require product-specific analytical reagents such as monoclonal antibodies. A MS-based capsid identity test is suitable for cGMP lot release testing of rAAV gene transfer products and will help ensure patient protection.
authors.affiliation: Department of Molecular Genetics and Microbiology, University of Florida, College of Medicine, United States.
authors.forename: Kim
authors.initials: K
authors.lastname: Van Vliet
authors.forename: Yasmin
authors.initials: Y
authors.lastname: Mohiuddin
authors.forename: Scott
authors.initials: S
authors.lastname: McClung
authors.forename: Veronique
authors.initials: V
authors.lastname: Blouin
authors.forename: Fabienne
authors.initials: F
authors.lastname: Rolling
authors.forename: Philippe
authors.initials: P
authors.lastname: Moullier
authors.forename: Mavis
authors.initials: M
authors.lastname: Agbandje-McKenna
authors.forename: Richard O
authors.initials: RO
authors.lastname: Snyder
mesh_terms: D036022:Capsid Proteins / Q000737:chemistry*; D002460:Cell Line; D000229:Dependovirus / Q000737:chemistry* / Q000145:classification*; D005822:Genetic Vectors / Q000145:classification; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D012680:Sensitivity and Specificity; D012703:Serotyping
chemical_list: D036022:Capsid Proteins