Purification of Recombinant Adeno-Associated Virus Vectors by Column Chromatography and Its Performancein Vivo

Abstract

Recombinant adeno-associated virus (AAV) holds much promise for human gene therapy. While evidence indicates that AAV mediates long-term gene transfer in several different tissues, difficulty in preparing and purifying this viral vector in large quantities remains a major obstacle for evaluating AAV vectors in clinical trials. The current method of purification, based on sedimentation through cesium chloride, is not scaleable and yields product of insufficient quality. In this article we report a new technique for purifying AAV, using a fully closed two-column chromatography system. Yields of AAV vectors purified by this method are high, potency is increased, and the purity of column-purified preparations is substantially improved. We previously reported a novel method to generate AAV based on an AAV Rep/Cap-containing cell line (B50) and an Ad-AAV hybrid virus, which is amenable to scale-up in bioreactors. By combining the new, fully scaleable purification process we report here with the B50/hybrid production method, it would be feasible to prepare AAV vectors to the scale and purity required for clinical and potential commercial applications.

Chromatographic purification of recombinant adenoviral and adeno-associated viral vectors: methods and implications

“…[34][35][36] Generation of stable packaging cell lines expressing high levels of Rep and Cap proteins is complicated by the well-documented strong cytostatic effect of the AAV Rep protein. 37,38 Stable packaging cell lines were created from HeLa cells by integration of the native AAV genome including a Rep-Cap gene cassette, but without both ITR sequences.…”

“…46 In addition to a nuclease, some purification methods require detergents and protease treatment. 20,36,[46][47][48][49][50] Some of these additives are hard to eliminate and therefore cannot be used for commercial manufacturing. In contrast to Ad, the relative stability of the AAV particle to heat, mild proteolytic digestion and nonionic detergents allows it to withstand robust purification procedures, thus facilitating scaled-up production.…”

“…Purification of AAV2, AAV4 and AAV5 vectors using ion-exchange resins has been reported by several groups. 36,44,54,55 AAV2 binding to cation-exchange resin at a physiological pH can be utilized as a first purification step. The AAV packaging system based on the Rep-Cap cell line B50 and an Ad-AAV hybrid virus was chosen to establish a robust, scaleable column purification process for the AAV2 vector suitable for commercial applications.…”

“…The AAV packaging system based on the Rep-Cap cell line B50 and an Ad-AAV hybrid virus was chosen to establish a robust, scaleable column purification process for the AAV2 vector suitable for commercial applications. 36 The authors used a POROS 20 HE (PerSeptive Biosystems, Framingham, MA, USA) cation-exchange column for the first separation step and a POROS 50 PI (PerSeptive Biosystems) anion-exchange column for the polishing step. AAV bound to the first column was eluted with 400 mM NaCl directly onto the POROS 50 PI column.…”

High AAV vector purity results in serotype- and tissue-independent enhancement of transduction efficiency

“…[1][2][3][4][5][6][7] An early challenge for AAV production and purification was to obtain sufficient amounts of high-titer vectors to support research studies in animal models. With steady advances in the field into clinical studies, and the isolation of several novel AAV serotypes, 8 more efficient and versatile production and purification methods have been required.…”