title: Comparative Analysis of Cesium Chloride- and Iodixanol-Based Purification of Recombinant Adeno-Associated Viral Vectors for Preclinical Applications.
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.
authors.affiliation: 1 Target Discovery Research, Boehringer Ingelheim Pharma GmbH & Co. KG , Biberach an der Riss, Germany .
authors.forename: Benjamin
authors.initials: B
authors.lastname: Strobel
authors.affiliation: 1 Target Discovery Research, Boehringer Ingelheim Pharma GmbH & Co. KG , Biberach an der Riss, Germany .
authors.forename: Felix D
authors.initials: FD
authors.lastname: Miller
authors.affiliation: 2 Drug Discovery Support, Boehringer Ingelheim Pharma GmbH & Co. KG , Biberach an der Riss, Germany .
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Rist
authors.affiliation: 1 Target Discovery Research, Boehringer Ingelheim Pharma GmbH & Co. KG , Biberach an der Riss, Germany .
authors.forename: Thorsten
authors.initials: T
authors.lastname: Lamla
mesh_terms: D002499:Centrifugation, Density Gradient / Q000379:methods*; D002586:Cesium; D002712:Chlorides; D002850:Chromatography, Gel / Q000379:methods; D000229:Dependovirus / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics* / Q000302:isolation & purification* / Q000648:ultrastructure; D057809:HEK293 Cells; D006801:Humans; D011092:Polyethylene Glycols; D014283:Triiodobenzoic Acids
chemical_list: D002712:Chlorides; D014283:Triiodobenzoic Acids; D002586:Cesium; D011092:Polyethylene Glycols; C028019:cesium chloride; C044834:iodixanol