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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

Analysis of the fractionated iodixanol density gradient. One-milliliter fractions were collected from the bottom of the ultracentrifugation tube and analyzed for bioactive AAV particles and contaminants analogous to the description in Fig. 2. Color image available online at www.liebertpub.com/hgtb
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f3: Analysis of the fractionated iodixanol density gradient. One-milliliter fractions were collected from the bottom of the ultracentrifugation tube and analyzed for bioactive AAV particles and contaminants analogous to the description in Fig. 2. Color image available online at www.liebertpub.com/hgtb

Mentions: Our results show that AAV vector genomes could be detected mainly in fractions 6–11, with additional CMV-positive sequences also measured up to fraction 27 (Fig. 3a). Plasmid DNA was measured mainly in fractions 5–7 and 12–27 (Fig. 3a), whereas genomic DNA was present solely in fractions 12–27. AAV8 capsids were detected in fractions 5–13 (Fig. 3b). Notably, in contrast to the CsCl gradient, where two peaks were obtained, here just one main peak around fraction 7 was present; however, this peak showed a “shoulder” in fractions 12 and 13 (Fig. 3b). We next analyzed the fractions for bioactive AAVs and obtained GFP-positive HEK-293 cells mainly when treated with fractions 5–11, whereas fractions 12–18 and 24–32 showed only some low-level signal (Fig. 3b). Finally, we analyzed the fractions for protein impurities. Our SDS-PAGE results show that VP1, VP2, and VP3 could be clearly identified in fractions 5–10, whereas massive protein contaminations were obtained in fractions 11 and 12 (Fig. 3c). The presence of contaminating proteins was also evident from an increase in absorption at 340 nm (Supplementary Fig. S3), which was previously shown to be a suitable measuring method to separate AAV-containing fractions from the major protein contaminations in the upper fractions of the 40% phase.5 In addition, a drop in the RI can serve as a further indicator of iodixanol phase changes (Supplementary Fig. S4). Taken together, to obtain bioactive AAV particles with the lowest impurities possible, fractions 6–10 should be isolated.


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)

Analysis of the fractionated iodixanol density gradient. One-milliliter fractions were collected from the bottom of the ultracentrifugation tube and analyzed for bioactive AAV particles and contaminants analogous to the description in Fig. 2. Color image available online at www.liebertpub.com/hgtb
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Analysis of the fractionated iodixanol density gradient. One-milliliter fractions were collected from the bottom of the ultracentrifugation tube and analyzed for bioactive AAV particles and contaminants analogous to the description in Fig. 2. Color image available online at www.liebertpub.com/hgtb
Mentions: Our results show that AAV vector genomes could be detected mainly in fractions 6–11, with additional CMV-positive sequences also measured up to fraction 27 (Fig. 3a). Plasmid DNA was measured mainly in fractions 5–7 and 12–27 (Fig. 3a), whereas genomic DNA was present solely in fractions 12–27. AAV8 capsids were detected in fractions 5–13 (Fig. 3b). Notably, in contrast to the CsCl gradient, where two peaks were obtained, here just one main peak around fraction 7 was present; however, this peak showed a “shoulder” in fractions 12 and 13 (Fig. 3b). We next analyzed the fractions for bioactive AAVs and obtained GFP-positive HEK-293 cells mainly when treated with fractions 5–11, whereas fractions 12–18 and 24–32 showed only some low-level signal (Fig. 3b). Finally, we analyzed the fractions for protein impurities. Our SDS-PAGE results show that VP1, VP2, and VP3 could be clearly identified in fractions 5–10, whereas massive protein contaminations were obtained in fractions 11 and 12 (Fig. 3c). The presence of contaminating proteins was also evident from an increase in absorption at 340 nm (Supplementary Fig. S3), which was previously shown to be a suitable measuring method to separate AAV-containing fractions from the major protein contaminations in the upper fractions of the 40% phase.5 In addition, a drop in the RI can serve as a further indicator of iodixanol phase changes (Supplementary Fig. S4). Taken together, to obtain bioactive AAV particles with the lowest impurities possible, fractions 6–10 should be isolated.

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