Abstract
Endotoxin is the most common contaminant found in protein samples. Even a small amount of endotoxin can induce strong allergic reaction and death of a host organism. Endotoxin is also often detected in recombinant adeno-associated virus (rAAV) stocks prepared in research laboratories using off-the-shelf reagents; purifying rAAV stocks from endotoxin using commercial reagents sometimes results in significant titer loss. The problem is exacerbated due to the recently expanded diversity of rAAV serotypes and capsid variants, which, due to their variable capsid surface charge, display differential affinity toward endotoxin. In this paper, we describe a simple universal protocol of purifying vector stocks irrespective of AAV serotype. The protocol is based on subjecting endotoxin-contaminated rAAV to mild detergent treatment, followed by repeated buffer-exchange washing and concentrating viral stock by low-speed centrifugation. Multiple assays were employed to test the physical and biological equivalency of the viral stocks before and after purification. The described protocol has been routinely utilized to purify vector stocks contaminated at levels as high as >1,000 endotoxin units (EU)/mL to produce viral vectors with practically undetectable levels of endotoxin (<2.5 EU/mL), with the titer’s recovery in the range of 50%–100%.
Highlights
Endotoxin is a lipopolysaccharide (LPS) present in the outer membrane of Gram-negative bacteria
At the physiological pH range of pH 7–8, the clusters of positively charged basic amino acid residues provide sufficient substrate for electrostatic interactions of capsid proteins with negatively charged LPS molecules, either aggregated or monomers. This interaction is enhanced by micelle-stabilizing divalent cations (Ca2+ and Mg2+), presenting a technical challenge when attempting to separate LPS and recombinant adeno-associated virus (rAAV)
We describe a protocol that reduces LPS level in rAAV preparations to practically undetectable levels (
Summary
Endotoxin is a lipopolysaccharide (LPS) present in the outer membrane of Gram-negative bacteria. Cell activation by LPS triggers the clinical syndrome of Gram-negative sepsis that can lead to fever, hypotension, respiratory and renal failure, and intravascular disseminated coagulation. These reactions are induced upon intravenous injection of LPS at concentrations as low as 1 ng/mL, which is equivalent to about 10 LPS units (endotoxin units [EU]).[1] according to US Food and Drug Administration (FDA) guidelines, the levels of LPSs in intravenously injected biopharmaceuticals must not exceed 5 EU/1 kg body weight.[2]
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