Universal Method for the Purification of Recombinant AAV Vectors of Differing Serotypes

Abstract

The generation of clinical good manufacturing practices (GMP)-grade adeno-associated virus (AAV) vectors requires purification strategies that support the generation of vectors of high purity, and that exhibit a good safety and efficacy profile. To date, most reported purification schemas are serotype dependent, requiring method development for each AAV gene therapy product. Here, we describe a platform purification process that is compatible with the purification of multiple AAV serotypes. The method generates vector preparations of high purity that are enriched for capsids with full vector genomes, and that minimizes the fractional content of empty capsids. The two-column purification method, a combination of affinity and ion exchange chromatographies, is compatible with a range of AAV serotypes generated by either the transient triple transfection method or the more scalable producer cell line platform. In summary, the adaptable purification method described can be used for the production of a variety of high-quality AAV vectors suitable for preclinical testing in animal models of diseases.

Extensive Transduction and Enhanced Spread of a Modified AAV2 Capsid in the Non-human Primate CNS

“…Purification of the vector was performed by column chromatography as previously described. 46 The vector preparation was substantially free of contaminants, as assessed by SDS-PAGE analysis with SYPRO staining; additionally the fractional content of empty capsids in the vector preparation was low at 13%, as determined by analytical ultracentrifugation ( Figure S4). 47…”

Moving from the bench towards a large scale, industrial platform process for adeno‐associated viral vector purification

“…Triton X-100 is a commonly used detergent that has shown to be effective yet inexpensive. Use of detergents is concentration and time dependent, with reported concentrations of Triton X-100 between 0.1% (2 hr and 37°C; Nass et al, 2018) it poses a potential concern for the downstream impact on chromatographic unit operations exists. The use of acidic buffers for cell lysis, such as citrate at pH 4.2, has also been explored (Kimura et al, 2019;Shuhei Sakamoto et al, 2018) and could prove an alternative to detergents that could mitigate the impact on downstream unit operations as well as the need to show clearance of the additive.…”

“…The first commercially available camelid resin was AVB Sephar-ose™ High Performance (GE Healthcare Life Sciences, 2016) which combines an agarose (Sepharose) base bead with a ligand that was isolated from llamas naturally exposed to wild-type AAV (Nass et al, 2018). As such, during development of this resin it was found that the antibody fragment selected displayed high affinity for AAV vectors with capsids of serotypes 1, 2, 3, and 5 (GE Healthcare Life Sciences, 2016).…”

Intrinsic Differential Scanning Fluorimetry for Fast and Easy Identification of Adeno-Associated Virus Serotypes

“…However, the differences were too small to clearly distinguish between full and empty AAVs in a complex sample mixture. For this purpose, other analytical methods can be used such as anion-exchange-chromatography, 33,34 analytical ultracentrifugation, 35,36 and transmission-or cryo-electron microscopy. 37 In summary, DSF offers a rapid, cost-effective, and robust method for AAV serotype identification due to their unique thermal stability profiles in the commonly utilized formulation and storage buffers and detergents.…”