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

Strobel B, Miller FD, Rist W, Lamla T - Hum Gene Ther Methods (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: 1 Target Discovery Research, Boehringer Ingelheim Pharma GmbH & Co. KG , Biberach an der Riss, Germany .

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.

No MeSH data available.


Related in: MedlinePlus

Process steps and duration of CsCl- and iodixanol-based adeno-associated virus (AAV) purification. After cell lysis by three repeated freeze–thaw cycles, the AAV-containing lysate is purified by either a CsCl- or iodixanol-based purification process. CsCl: Nucleic acids and proteins are pelleted by CaCl2 and PEG-8000 precipitation, respectively. After protein pellet dissolving overnight (o/n) and subsequent CsCl density gradient ultracentrifugation, CsCl is removed from the target fractions by repeated dialysis cycles. The AAV suspension is finally concentrated by ultrafiltration and sterile filtered. Iodixanol: The cell lysate is applied to iodixanol density gradient ultracentrifugation. Iodixanol is removed by three repeated ultrafiltration/concentration steps and the AAV suspension is finally sterile filtered.
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f1: Process steps and duration of CsCl- and iodixanol-based adeno-associated virus (AAV) purification. After cell lysis by three repeated freeze–thaw cycles, the AAV-containing lysate is purified by either a CsCl- or iodixanol-based purification process. CsCl: Nucleic acids and proteins are pelleted by CaCl2 and PEG-8000 precipitation, respectively. After protein pellet dissolving overnight (o/n) and subsequent CsCl density gradient ultracentrifugation, CsCl is removed from the target fractions by repeated dialysis cycles. The AAV suspension is finally concentrated by ultrafiltration and sterile filtered. Iodixanol: The cell lysate is applied to iodixanol density gradient ultracentrifugation. Iodixanol is removed by three repeated ultrafiltration/concentration steps and the AAV suspension is finally sterile filtered.

Mentions: Recombinant adeno-associated viral (AAV) vectors have evolved as one of the most heavily used vector tools to study gene function both in vitro and in vivo. Besides the fact that AAVs are regarded as nonpathogenic, one major advantage of AAVs is the lack of inflammation after vector application in vivo,1 thereby lowering the risk of altering experimental readouts by vector-induced immunogenic effects. In this regard, high purity of vector preparations is a key goal of AAV manufacturing both for research and clinical applications. For the serotype-independent purification of AAV vectors produced in HEK-293 or SF9 cells, the two most widely established protocols for preclinical applications are based on ultracentrifugation using either a continuous cesium chloride (CsCl) density gradient2 or an iodixanol step density gradient.3 While the CsCl protocol contains multiple purification steps (cell lysis, precipitation of DNA and proteins, ultracentrifugation, dialysis, concentration; see Fig. 1), rendering it a rather time-consuming (∼3.5 days) protocol, the iodixanol protocol requires only cell lysis, 2 hr of ultracentrifugation, and concentration, allowing for vector purification in 1 day (Fig. 1).


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

Strobel B, Miller FD, Rist W, Lamla T - Hum Gene Ther Methods (2015)

Process steps and duration of CsCl- and iodixanol-based adeno-associated virus (AAV) purification. After cell lysis by three repeated freeze–thaw cycles, the AAV-containing lysate is purified by either a CsCl- or iodixanol-based purification process. CsCl: Nucleic acids and proteins are pelleted by CaCl2 and PEG-8000 precipitation, respectively. After protein pellet dissolving overnight (o/n) and subsequent CsCl density gradient ultracentrifugation, CsCl is removed from the target fractions by repeated dialysis cycles. The AAV suspension is finally concentrated by ultrafiltration and sterile filtered. Iodixanol: The cell lysate is applied to iodixanol density gradient ultracentrifugation. Iodixanol is removed by three repeated ultrafiltration/concentration steps and the AAV suspension is finally sterile filtered.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4554548&req=5

f1: Process steps and duration of CsCl- and iodixanol-based adeno-associated virus (AAV) purification. After cell lysis by three repeated freeze–thaw cycles, the AAV-containing lysate is purified by either a CsCl- or iodixanol-based purification process. CsCl: Nucleic acids and proteins are pelleted by CaCl2 and PEG-8000 precipitation, respectively. After protein pellet dissolving overnight (o/n) and subsequent CsCl density gradient ultracentrifugation, CsCl is removed from the target fractions by repeated dialysis cycles. The AAV suspension is finally concentrated by ultrafiltration and sterile filtered. Iodixanol: The cell lysate is applied to iodixanol density gradient ultracentrifugation. Iodixanol is removed by three repeated ultrafiltration/concentration steps and the AAV suspension is finally sterile filtered.
Mentions: Recombinant adeno-associated viral (AAV) vectors have evolved as one of the most heavily used vector tools to study gene function both in vitro and in vivo. Besides the fact that AAVs are regarded as nonpathogenic, one major advantage of AAVs is the lack of inflammation after vector application in vivo,1 thereby lowering the risk of altering experimental readouts by vector-induced immunogenic effects. In this regard, high purity of vector preparations is a key goal of AAV manufacturing both for research and clinical applications. For the serotype-independent purification of AAV vectors produced in HEK-293 or SF9 cells, the two most widely established protocols for preclinical applications are based on ultracentrifugation using either a continuous cesium chloride (CsCl) density gradient2 or an iodixanol step density gradient.3 While the CsCl protocol contains multiple purification steps (cell lysis, precipitation of DNA and proteins, ultracentrifugation, dialysis, concentration; see Fig. 1), rendering it a rather time-consuming (∼3.5 days) protocol, the iodixanol protocol requires only cell lysis, 2 hr of ultracentrifugation, and concentration, allowing for vector purification in 1 day (Fig. 1).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: 1 Target Discovery Research, Boehringer Ingelheim Pharma GmbH & Co. KG , Biberach an der Riss, Germany .

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.

No MeSH data available.


Related in: MedlinePlus