508. Development of Optimized AAV2 Vector Biosynthesis and Purification Processes To Support High Capacity Production of Clinical Vectors of High Purity and Potency

Abstract

With the objective of establishing a capability to produce clinical grade gene transfer vectors, we have developed and implemented a novel AAV2 vector manufacturing process that enables scalable production and purification of AAV vectors of high purity, potency and safety. Biosynthesis of vector particles is achieved using transient transfection of HEK293 cells in the absence of helper virus. Our helper and packaging plasmids, in conjuction with an optimized transfection protocol, enable biosynthesis of approx. 100,000 vg per cell. The complete harvest (cells and media) is concentrated 5- fold and diafiltered by tangential flow filtration using a 100kDa NMWC membrane. Following cell lysis and clarification, AAV2 particles (vectors and empty particles) are separated from host cellular impurities by cation exchange chromatography. Highly efficient nuclease digestion is achieved by adding Benzonase to the column bound vector under conditions optimized to remove residual nucleic acids. This step was found to be highly effective for the removal of trace levels of vector-associated nucleic acids (host cellular and residual plasmid DNA) implicated in aggregation of subsequently purified vector particles. The eluate from the cation exchange resin is then subjected to a single gradient CsCl ultra-centrifugation step to separate empty capsids from the vector particles. In light of recent studies that AAV2 capsid protein contains epitopes recognized by cytotoxic T cells in a hemophilia B clinical study (Manno et al., Nat. Med. 2006), we have included the gradient step because we believe that removal of excess viral capsids reduces the potential for vector immunogenicity. A single gradient ultracentrifugation using laboratory scale equipment is sufficient to process (ie. remove empty capsid from) up to 1.0 E15 vg per centrifugation run, providing sufficient capacity to support clinical trials for current applications. A series of AAV2-hFIX vectors lots produced using this process gave a high degree of consistency in purity as assessed by SDS-PAGE silver staining analysis and potency as assessed in vitro by transduction of HepG2 cells, and in vivo by tail vein injection in C57/B6 mice. In comparison studies, the combined column / CsCl gradient purified vector demonstrated potency in vivo equal to or superior to vector purified by gradient ultracentrifugation or column chromatography alone. This combination of process steps has enabled us to initiate clinical vector production of AAV2 vectors of high safety, purity, and potency and at a capacity to meet the requirements of a range of clinical studies.