90. Identification of an Adeno-Associated Virus Binding Epitope for AVB Sepharose Affinity Resin

Abstract

Antibody-based affinity chromatography is increasingly becoming an important technology in clinical AAV vector production due to the one-step removal of the large majority of host cell contaminants and simplification of downstream processes. The affinity resin AVB Sepharose (GE) is widely used in AAV purification but has a major drawback in that it does not bind all serotypes of AAV equally well. Here we report the identification of an epitope, SPAKFA, of adeno-associated virus 3B (AAV3B) which specifically mediates binding to AVB Sepharose resin. Substitution of AAV3B SPAKFA for the corresponding peptide in two poorly-binding AAV serotypes, AAV8 and rh.64R1, resulted in greatly increased affinity to AVB sepharose. In addition, the capacity of AVB Sepharose for the AAV9 serotype, which binds extremely poorly in the context of the naturally occurring epitope, could be augmented substantially upon replacement with SPAKFA. Importantly, the substitution of the AAV3B SPAKFA peptide did not affect the potency of the serotypes tested in an in vitro assay. The sequence analysis -resin binding combination approach used here to identify the SPAKFA peptide may prove to be a powerful resin selection tool for novel AAV serotypes. In addition, our discovery suggests the possibility of vector engineering to yield a universal method for the purification of multiple AAV serotypes.