Removal of Empty Capsids from Type 1 Adeno-Associated Virus Vector Stocks by Anion-Exchange Chromatography Potentiates Transgene Expression

Abstract

Production of recombinant adeno-associated virus (rAAV) results in substantial quantities of empty capsids or virus-like particles (VLPs), virus protein shells without the vector genome. The contaminating VLPs would interfere with transduction by competing for cell-surface receptors and, when administered in vivo, contribute to antigen load, which may elicit a stronger immune response. Density-gradient ultracentrifugation provides a means to separate VLPs from rAAV particles, but is not feasible for large-scale preparations of vectors. Since the compositions of the VLP and vector differ by the single-stranded DNA genome, we hypothesized that the isoelectric point of the vector may differ from that of the VLP. In an attempt to separate type 1 rAAV particles from VLPs by ion-exchange chromatography, we tested a number of buffer systems and found that trimethylammonium sulfate, or [(CH3)4N]2SO4, effectively separated rAAV1 particles from VLPs. The rAAV1-GFP chromatographically separated from VLPs induced stronger GFP expression in HEK293 cells than rAAV1-GFP contaminated with VLPs. The transduction of mouse muscles with rAAV1-SEAP (secreted form of alkaline phosphatase) isolated from VLPs also showed higher serum SEAP levels than rAAV1-SEAP with VLPs. These results suggest that chromatographic separation of rAAV1 from empty capsids increased the efficacy of rAAV1.

Adeno-associated virus capsid antigen presentation is dependent on endosomal escape

“…During AAV production, a critical concern for clinical trials is the contamination of vector particles lacking a genome, especially when using purification approaches with column chromatography (45,46). These empty virions substantially increase the dose of AAV capsid proteins and possibly result in an unwanted immunological response and decreased transgenic expression.…”

“…AAV vectors purified from CsCl have been applied in clinical trials, however this purification approach is not scalable. Recently, ion exchange chromatography has been studied to purify AAV vectors (43)(44)(45)(46). Unlike the CsCl purification approach, the chromatographic method cannot currently separate genome-containing particles of AAV vectors (full particles) from empty virions because the virion surfaces are the same.…”

“…Unlike the CsCl purification approach, the chromatographic method cannot currently separate genome-containing particles of AAV vectors (full particles) from empty virions because the virion surfaces are the same. The contamination of empty virions inhibits transduction from full particles of AAV vectors and potentially increases the virus capsid antigen load in transduced cells (46). To investigate the possibility of antigen presentation by empty particles, we made empty AAV vector by transfecting only 2 plasmids (pXX6-80 and pXR2OVA) instead of using 3 plasmids for AAV vector production.…”

AAV2/8 Vectors Purified from Culture Medium with a Simple and Rapid Protocol Transduce Murine Liver, Muscle, and Retina Efficiently

“…Iodixanol ultra-high-speed density gradient centrifugation (Hermens et al, 1999;Zolotukhin et al, 1999) is similar to CsCl 2 ultracentrifugation, but with higher vector recovery yields although expensive and relatively time-consuming. The majority of chromatographic purification methods use either ion-exchange (Urabe et al, 2006;Qu et al, 2007;Okada et al, 2009) or affinity-based techniques (Auricchio et al, 2001;Brument et al, 2002;Kaludov et al, 2002;Zolotukhin et al, 2002) to purify AAV. The latter use either antibodies directed to the assembled AAV capsid, which are mostly serotype specific (Anderson et al, 2000), or heparin/sialic acid-based affinities, which are based on the property of some AAV serotypes (i.e., AAV2/2 and AAV2/5), to bind with high-affinity heparin sulfate proteoglycan (Summerford and Samulski, 1998) or sialic acid, respectively (Walters et al, 2001).…”

Analysis of Particle Content of Recombinant Adeno-Associated Virus Serotype 8 Vectors by Ion-Exchange Chromatography

“…Although effective, such methods are not suitable to cope with the vector production demands of late-stage clinical trials and commercial manufacturing and more scalable processes are required. Scalable methods of empty particle removal have been described whereby the separation of empty and full AAV vector particles of serotypes 1, 2, and 6 was achieved on the basis of charge, using ion-exchange chromatography (Urabe et al, 2006;Qu et al, 2007;Okada et al, 2009). This type of approach is compatible with large-scale AAV vector production and if sufficiently robust, is likely to supplant gradient-based separation technologies in commercial manufacturing.…”