Recombinant Adeno-associated virus (rAAV) is extensively used as a vector for gene therapy/DNA vaccine delivery in a multitude of ongoing clinical trials. Consequently, development of protocols for manufacturing of a clinical grade rAAV free of contaminants becomes a high priority in industrial as well as research laboratory setting.<br/>One of the most pervasive but frequently overlooked impurities in rAAV preparations is endotoxin. Endotoxins (Lipopolysaccharides, LPS) are part of outer membrane in Gram-negative bacteria and, as a contaminant in the injection sample, can induce an acute inflammatory response in a trial subject or in animal model.<br/>In the current report, we describe a simple, affordable, and serotype-independent protocol for the purification of rAAV preparations contaminated with endotoxin. The protocol is based on the disruption of LPS-rAAV capsid complexes by mild non-ionic detergent treatment with subsequent physical separation of LPS monomers using centrifugal filter concentrator, followed by buffer exchange to remove detergent, and virus concentration under conditions avoiding virus aggregation. The quality LPS-purified rAAVs were assessed using physical and biological tests to establish the equivalency of samples during the purification procedure.<br/>Described method reduces endotoxin in rAAV samples from more than 1000 EU/mL to &lt;2.5 with titer recovery 50-90% and no noticeable change in virus infectivity.

Development of a simple protocol of purifying rAAV vectors from endotoxin contamination

2019 Spring National Meeting

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