title: Analytical Ultracentrifugation as an Approach to Characterize Recombinant Adeno-Associated Viral Vectors.
abstract: Recombinant adeno-associated viral (rAAV) vectors represent a novel class of biopharmaceutical drugs. The production of clinical-grade rAAV vectors for gene therapy would benefit from analytical methods that are able to monitor drug product quality with regard to homogeneity, purity, and manufacturing consistency. Here, we demonstrate the novel application of analytical ultracentrifugation (AUC) to characterize the homogeneity of preparations of rAAV vectors. We show that a single sedimentation velocity run of rAAV vectors detected and quantified a number of different viral species, such as vectors harboring an intact genome, lacking a vector genome (empty particles), and containing fragmented or incomplete vector genomes. This information is obtained by direct boundary modeling of the AUC data generated from refractometric or UV detection systems using the computer program SEDFIT. Using AUC, we show that multiple parameters contributed to vector quality, including the AAV genome form (i.e., self-complementary vs. single-stranded), vector genome size, and the production and purification methods. Hence, AUC is a critical tool for identifying optimal production and purification processes and for monitoring the physical attributes of rAAV vectors to ensure their quality.
authors.affiliation: Gene Therapy, Genzyme, a Sanofi Company , Framingham, Massachusetts.
authors.forename: Brenda
authors.initials: B
authors.lastname: Burnham
authors.affiliation: Gene Therapy, Genzyme, a Sanofi Company , Framingham, Massachusetts.
authors.forename: Shelley
authors.initials: S
authors.lastname: Nass
authors.affiliation: Gene Therapy, Genzyme, a Sanofi Company , Framingham, Massachusetts.
authors.forename: Elton
authors.initials: E
authors.lastname: Kong
authors.affiliation: Gene Therapy, Genzyme, a Sanofi Company , Framingham, Massachusetts.
authors.forename: MaryEllen
authors.initials: M
authors.lastname: Mattingly
authors.affiliation: Gene Therapy, Genzyme, a Sanofi Company , Framingham, Massachusetts.
authors.forename: Denise
authors.initials: D
authors.lastname: Woodcock
authors.affiliation: Gene Therapy, Genzyme, a Sanofi Company , Framingham, Massachusetts.
authors.forename: Antonius
authors.initials: A
authors.lastname: Song
authors.affiliation: Gene Therapy, Genzyme, a Sanofi Company , Framingham, Massachusetts.
authors.forename: Samuel
authors.initials: S
authors.lastname: Wadsworth
authors.affiliation: Gene Therapy, Genzyme, a Sanofi Company , Framingham, Massachusetts.
authors.forename: Seng H
authors.initials: SH
authors.lastname: Cheng
authors.affiliation: Gene Therapy, Genzyme, a Sanofi Company , Framingham, Massachusetts.
authors.forename: Abraham
authors.initials: A
authors.lastname: Scaria
authors.affiliation: Gene Therapy, Genzyme, a Sanofi Company , Framingham, Massachusetts.
authors.forename: Catherine R
authors.initials: CR
authors.lastname: O'Riordan
mesh_terms: D018929:Cell Culture Techniques; D002460:Cell Line; D002852:Chromatography, Ion Exchange / Q000379:methods; D000229:Dependovirus / Q000235:genetics*; D015870:Gene Expression; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D010957:Plasmids / Q000235:genetics; D014161:Transduction, Genetic; D019076:Transgenes; D014461:Ultracentrifugation / Q000379:methods* / Q000592:standards; D014779:Virus Replication