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

Abstract

Advances in adeno-associated virus (AAV)-mediated gene therapy have brought the possibility of commercial manufacturing of AAV vectors one step closer. To realize this prospect, a parallel effort with the goal of ever-increasing sophistication for AAV vector production technology and supporting assays will be required. Among the important release assays for a clinical gene therapy product, those monitoring potentially hazardous contaminants are most critical for patient safety. A prominent contaminant in many AAV vector preparations is vector particles lacking a genome, which can substantially increase the dose of AAV capsid proteins and lead to possible unwanted immunological consequences. Current methods to determine empty particle content suffer from inconsistency, are adversely affected by contaminants, or are not applicable to all serotypes. Here we describe the development of an ion-exchange chromatography-based assay that permits the rapid separation and relative quantification of AAV8 empty and full vector particles through the application of shallow gradients and a strong anion-exchange monolith chromatography medium.

Absolute Determination of Single-Stranded and Self-Complementary Adeno-Associated Viral Vector Genome Titers by Droplet Digital PCR

“…All vector preparations were prepared by triple transfection of HEK293 cells followed by purification by either the iodixanol gradient method as described by Lock and colleagues (2010a) or over CsCl gradients as described previously (Lock et al, 2012).…”

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

“…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.…”

Downstream processing and chromatography based analytical methods for production of vaccines, gene therapy vectors, and bacteriophages

“…Due to the large void volume of membranes compared to monoliths, the resolution, regardless of the flow rate used, is superior with monoliths. This advantage of monoliths is especially important and has already been recognized in different DSP steps of nanoparticles [6,8,9,10,11]. Excellent resolution of monoliths was demonstrated by Lock's group [9] who developed a method for separation of two very similar viral particles (viral capsids with and without genetic material) using CIM monolithic column and a very shallow linear gradient.…”