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Comparative Analysis of Cesium Chloride- And Iodixanol-Based Purification of Recombinant Adeno-Associated Viral Vectors for Preclinical Applications

Comparative Analysis of Cesium Chloride- And Iodixanol-Based Purification of Recombinant Adeno-Associated Viral Vectors for Preclinical Applications

Benjamin Strobel, Felix D Miller, Wolfgang Rist, Thorsten Lamla

Hum Gene Ther Methods. 2015 Aug;26(4):147-57.

PMID: 26222983

Abstract:

Cesium chloride (CsCl)- and iodixanol-based density gradients represent the core step in most protocols for serotype-independent adeno-associated virus (AAV) purification established to date. However, despite controversial reports about the purity and bioactivity of AAV vectors derived from each of these protocols, systematic comparisons of state-of-the-art variants of these methods are sparse. To define exact conditions for such a comparison, we first fractionated both gradients to analyze the distribution of intact, bioactive AAVs and contaminants, respectively. Moreover, we tested four different polishing methods (ultrafiltration, size-exclusion chromatography, hollow-fiber tangential flow filtration, and polyethylene glycol precipitation) implemented after the iodixanol gradient for their ability to deplete iodixanol and protein contaminations. Last, we conducted a side-by-side comparison of the CsCl and iodixanol/ultrafiltration protocol. Our results demonstrate that iodixanol-purified AAV preparations show higher vector purity but harbor more (∼20%) empty particles as compared with CsCl-purified vectors (<1%). Using mass spectrometry, we analyzed prominent protein impurities in the AAV vector product, thereby identifying known and new, possibly AAV-interacting proteins as major contaminants. Thus, our study not only provides a helpful guide for the many laboratories entering the AAV field, but also builds a basis for further investigation of cellular processes involved in AAV vector assembly and trafficking.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP7647178	Cesium chloride		7647-17-8	Price

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