title: Identification of an adeno-associated virus binding epitope for AVB sepharose affinity resin.
abstract: Recent successes of adeno-associated virus (AAV)-based gene therapy have created a demand for large-scale AAV vector manufacturing and purification techniques for use in clinical trials and beyond. During the development of purification protocols for rh.10, hu.37, AAV8, rh.64R1, AAV3B, and AAV9 vectors, based on a widely used affinity resin, AVB sepharose (GE), we found that, under the same conditions, different serotypes have different affinities to the resin, with AAV3B binding the best and AAV9 the poorest. Further analysis revealed a surface-exposed residue (amino acid number 665 in AAV8 VP1 numbering) differs between the high-affinity AAV serotypes (serine in AAV3B, rh.10, and hu.37) and the low-affinity ones (asparagine in AAV8, rh.64R1, and AAV9). The residue locates within a surface-exposed, variable epitope flanked by highly conserved residues. The substitution of the epitope in AAV8, rh.64R1, and AAV9 with the corresponding epitope of AAV3B (SPAKFA) resulted in greatly increased affinity to AVB sepharose with no reduction in the vectors' in vitro potency. The presence of the newly identified AVB-binding epitope will be useful for affinity resin selection for the purification of novel AAV serotypes. It also suggests the possibility of vector engineering to yield a universal affinity chromatography purification method for multiple AAV serotypes.
authors.affiliation: Gene Therapy Program, Department of Pathology and Laboratory Medicine, University of Pennsylvania Perelman School of Medicine , Philadelphia, Pennsylvania, USA.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Wang
authors.affiliation: Gene Therapy Program, Department of Pathology and Laboratory Medicine, University of Pennsylvania Perelman School of Medicine , Philadelphia, Pennsylvania, USA.
authors.forename: Martin
authors.initials: M
authors.lastname: Lock
authors.affiliation: Gene Therapy Program, Department of Pathology and Laboratory Medicine, University of Pennsylvania Perelman School of Medicine , Philadelphia, Pennsylvania, USA.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Prongay
authors.affiliation: Gene Therapy Program, Department of Pathology and Laboratory Medicine, University of Pennsylvania Perelman School of Medicine , Philadelphia, Pennsylvania, USA.
authors.forename: Mauricio R
authors.initials: MR
authors.lastname: Alvira
authors.affiliation: Gene Therapy Program, Department of Pathology and Laboratory Medicine, University of Pennsylvania Perelman School of Medicine , Philadelphia, Pennsylvania, USA.
authors.forename: Boris
authors.initials: B
authors.lastname: Petkov
authors.affiliation: Gene Therapy Program, Department of Pathology and Laboratory Medicine, University of Pennsylvania Perelman School of Medicine , Philadelphia, Pennsylvania, USA.
authors.forename: James M
authors.initials: JM
authors.lastname: Wilson