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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 protein impurities in HEK-293-derived AAV product preparations. Shown is a Coomassie blue-stained SDS-polyacrylamide gel of each of the final AAV preparations of batch 1 loaded at the indicated amounts. For batches 2 and 3, see Supplementary Fig. S6. Gel bands 1–13 were analyzed by mass spectrometry, and identified cellular proteins are presented in Table 1. M, marker lane. Color image available online at www.liebertpub.com/hgtb
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f6: Analysis of protein impurities in HEK-293-derived AAV product preparations. Shown is a Coomassie blue-stained SDS-polyacrylamide gel of each of the final AAV preparations of batch 1 loaded at the indicated amounts. For batches 2 and 3, see Supplementary Fig. S6. Gel bands 1–13 were analyzed by mass spectrometry, and identified cellular proteins are presented in Table 1. M, marker lane. Color image available online at www.liebertpub.com/hgtb

Mentions: Despite the relatively high degree of purity obtained with both CsCl- and iodixanol-based protocols, protein impurities became visible in both cases when higher AAV amounts were analyzed by SDS-PAGE (Fig. 5b and Supplementary Fig. S5). Because protein contamination in AAV vector preparations might cause immunological responses in vivo, which could potentially alter relevant experimental readouts, we attempted to identify the most prominent protein contamination in the final AAV preparations. For this purpose, we reran the final samples from our direct comparison of CsCl- and iodixanol-derived AAVs (Fig. 5b) on an SDS-polyacrylamide gel. After Coomassie blue staining, the most prominent bands (Fig. 6, indicated by numbers) were excised and analyzed by mass spectrometry. Notably, whereas bands 7–13 were present at similar intensity in CsCl- and iodixanol-purified preparations, bands 1–6 seemed to be present relatively specifically in the less pure CsCl AAV preparations (Fig. 6). A further point to note is that the band pattern was highly reproducible when comparing three independently produced and purified AAV batches (Supplementary Fig. S6).


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 protein impurities in HEK-293-derived AAV product preparations. Shown is a Coomassie blue-stained SDS-polyacrylamide gel of each of the final AAV preparations of batch 1 loaded at the indicated amounts. For batches 2 and 3, see Supplementary Fig. S6. Gel bands 1–13 were analyzed by mass spectrometry, and identified cellular proteins are presented in Table 1. M, marker lane. 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

f6: Analysis of protein impurities in HEK-293-derived AAV product preparations. Shown is a Coomassie blue-stained SDS-polyacrylamide gel of each of the final AAV preparations of batch 1 loaded at the indicated amounts. For batches 2 and 3, see Supplementary Fig. S6. Gel bands 1–13 were analyzed by mass spectrometry, and identified cellular proteins are presented in Table 1. M, marker lane. Color image available online at www.liebertpub.com/hgtb
Mentions: Despite the relatively high degree of purity obtained with both CsCl- and iodixanol-based protocols, protein impurities became visible in both cases when higher AAV amounts were analyzed by SDS-PAGE (Fig. 5b and Supplementary Fig. S5). Because protein contamination in AAV vector preparations might cause immunological responses in vivo, which could potentially alter relevant experimental readouts, we attempted to identify the most prominent protein contamination in the final AAV preparations. For this purpose, we reran the final samples from our direct comparison of CsCl- and iodixanol-derived AAVs (Fig. 5b) on an SDS-polyacrylamide gel. After Coomassie blue staining, the most prominent bands (Fig. 6, indicated by numbers) were excised and analyzed by mass spectrometry. Notably, whereas bands 7–13 were present at similar intensity in CsCl- and iodixanol-purified preparations, bands 1–6 seemed to be present relatively specifically in the less pure CsCl AAV preparations (Fig. 6). A further point to note is that the band pattern was highly reproducible when comparing three independently produced and purified AAV batches (Supplementary Fig. S6).

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