Journal of Virological Methods 139 (2007) 61–70 Primary recovery and chromatographic purification of adeno-associated virus type 2 produced by baculovirus/insect cell system Parminder S. Chahal, Marc G. Aucoin, Amine Kamen ∗ Animal Cell Technology Group, Bioprocess Sector, Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Avenue, Montreal, Quebec, Canada H4P2R2 Received 19 May 2006; received in revised form 14 September 2006; accepted 19 September 2006 Available online 20 October 2006 Abstract Adeno-associated virus (AAV) is making its place in gene therapy applications; however, the industry is still facing obstacles in producing a large quantity of highly purified material for clinical studies. Insect cell technology can be used to produce AAV to meet the current demand. During the purification process it was observed that there was a reduced recovery of AAV produced in insect cells, Spodoptera frugiperda (Sf9). It was assumed that the formation of AAV agglomerates and the interaction of AAV with other cellular components were major contributors to this loss. After studying different systems of extraction a sequence of treatment for primary recovery of AAV from cell paste was developed. This sequence was necessary to reduce the AAV losses and to increase the recovery. The purification method avoided the use of ultracentrifugation and adopted chromatographic methods for the purification of AAV. Primary recovery, ion exchange chromatography and hydrophobic interaction chromatography gave an overall yield of 75% from the extracted AAV. The purification process was based on chromatographic methods; therefore, it can be scaled up. Although this method was developed for AAV type 2, it is believed that this method could be modified easily to purify other AAV serotypes. Crown Copyright © 2006 Published by Elsevier B.V. All rights reserved. Keywords: AAV; Adeno-associated virus; Insect cell; Baculovirus; Purification; Primary recovery 1. Introduction Adeno-associated virus (AAV) is a non-pathogenic human parvovirus that requires helper functions for a productive infec- tion, generally in the form of co-infection by viruses, such as, adenovirus or herpes virus. Since AAV has become a leading candidate for gene therapy, there is a keen interest in the produc- tion of recombinant AAV vectors. The use of cloned plasmids, which avoids the use of intact helper virus, is a common practice (Samulski et al., 1982). An alternative method, which is also garnering interest, is the production of AAV vectors in insect cells, which avoids the use of any pathogenic helper components (Meghrous et al., 2005; Urabe et al., 2002). Although AAV has been considered a potential vector for gene therapy applications for more than a decade (Muzyczka, 1992), obstacles remain in ∗ Corresponding author. Tel.: +1 514 496 2264; fax: +1 514 496 6785. E-mail addresses: parminder.chahal@cnrc-nrc.gc.ca (P.S. Chahal), marc.aucoin@cnrc-nrc.gc.ca (M.G. Aucoin), amine.kamen@cnrc-nrc.gc.ca (A. Kamen). producing large quantities of highly purified material, which are required for clinical studies. There have been a number of approaches reported to purify AAV vectors. The conventional CsCl density gradient centrifu- gation has been adopted by many to purify AAV. This method, however, is time consuming and inefficient. Contact of AAV with CsCl for 24 to 72 h, which is normal for the CsCl gra- dient centrifugation method, has been reported to cause a loss of 50–87% of particle infectivity, respectively (Auricchio et al., 2001). The purity of AAV generated by this method is low and the potency obtained is reduced (Gao et al., 2000a). Above all, this method is not an easily scaleable process. Iodixanol, an alternative to CsCl, has shown encouraging results (Hermens et al., 1999; Zolotukhin et al., 1999, 2002) in terms of stability, recovery and short contact time. Although this process is quick, it is also limited to small-scale applications (Zolotukhin, 2005). Investigators have also explored chromatographic techniques to purify AAV (Anderson et al., 2000; Auricchio et al., 2001; Clark et al., 1999; Debelak et al., 2000; Gao et al., 2000a; Grimm et al., 1998; Hermens et al., 1999; Kaludov et al., 0166-0934/$ – see front matter. Crown Copyright © 2006 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2006.09.011