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.

Copackaged AAV9 Vectors Promote Simultaneous Immune Tolerance and Phenotypic Correction of Pompe Disease

“…51,88 The present study included, consistent reproducibility of the predicted packaging ratios in AAV2, AAV8, and AAV9 shows that the process is serotype-independent; production levels at surface areas ranging from 148 to 6,320 cm 2 using roller bottles or flasks highlight the scalability and flexibility in manufacturing; purification of copackaged vectors are not affected whether performing cesium-chloride or iodixanol centrifugation; and careful titration of the expression plasmids results in consistent ratios across preparations. 51,88,92 Rearrangement of the plasmid genome brought on by homologous recombination during packaging is a concern but seems to occur at a negligible rate as the output ratios are highly consistent of the predicted ratios. 51 Once the manufacturing process is defined, the copackaged candidate vector can therefore be treated as a single product for toxicology, stability, sterility, and release testing as performed for any current AAV clinical trial rather than as two independent products.…”

Utilizing minimally purified secreted rAAV for rapid and cost-effective manipulation of gene expression in the CNS

“…However, the widespread use of rAAV vectors is limited by the time and expense needed to produce them. The current methods for purifying rAAV utilize gradients of either iodixanol or cesium chloride [13][14][15][16][17]. These methods require the use of specialized centrifuges and expensive reagents which can prevent laboratories lacking the proper equipment or funding from producing rAAV in-house.…”

A generalized purification step for viral particles using mannitol flocculation

“…Ultracentrifugation is a popular choice for the purification of adeno‐associated virus (AAV) gene therapy vectors. A recent study showed a 60% yield for CsCl and a 65% infectious units recovery for an iodixanol based AAV purification method . While yield and purity can be high with ultracentrifugation, the lack of scale‐up options makes it unfeasible for long‐term, high volume production.…”

“…A recent study showed a 60% yield for CsCl and a 65% infectious units recovery for an iodixanol based AAV purification method. 5 While yield and purity can be high with ultracentrifugation, the lack of scale-up options makes it unfeasible for long-term, high volume production. A more scalable method of virus particle purification is ultrafiltration.…”