Abstract
We present membrane-based steric exclusion chromatography (SXC) as a universal capture step for purification of adeno-associated virus (AAV) gene transfer vectors independent of their serotype and surface characteristics. SXC is performed by mixing an unpurified cell culture supernatant containing AAV particles with polyethylene glycol (PEG) and feeding the mixture onto a chromatography filter unit. The purified AAV particles are recovered by flushing the unit with a solution lacking PEG. SXC is an inexpensive single-use method that permits to concentrate, purify, and re-buffer AAV particles with yields >95% and >80% impurity clearance. SXC could theoretically be employed at industrial scales with units of nearly 20 m2.
Highlights
ADENO-ASSOCIATED VIRUS (AAV)-based gene therapy offers the prospect of treating a wide range of diseases, such as cancer,[1,2] hemophilia,[3] Duchenne muscular dystrophy,[4] and vision loss.[5,6]
We focus on a purification method that is called ‘‘steric exclusion chromatography’’ (SXC) and that exploits molecular crowding effects caused by the addition of a ‘‘crowding agent’’ to a solution.[22]
We describe, for the first time, the use of membrane-based SXC as an efficient method for the purification of AAV vectors with self-made filter units packed with disposable cellulose membranes of 1.0 lm pore size
Summary
ADENO-ASSOCIATED VIRUS (AAV)-based gene therapy offers the prospect of treating a wide range of diseases, such as cancer,[1,2] hemophilia,[3] Duchenne muscular dystrophy,[4] and vision loss.[5,6] The promise and potential of this technology is perhaps best exemplified by the market authorization of three commercial gene therapy products derived from wild-type AAV serotypes, that is, Glybera (AAV1), LuxturnaÒ (AAV2) and, most recently, ZolgensmaÒ (AAV9). The removed cell supernatant contains AAV particles and can be directly processed as described in the ‘‘Sample Preparation for SXC’’ section for purification with SXC.
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