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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

Comparative analysis of polishing methods for AAVs isolated from an iodixanol gradient. AAV target fractions obtained from two parallel iodixanol gradients loaded with the HEK-293 cell lysate from a total of fifty 15-cm plates were further purified by ultrafiltration (UF), size-exclusion chromatography (SEC), hollow-fiber tangential flow filtration (HF-TFF), or PEG-8000 precipitation. (a) AAV vector yield and loss during purification by the various polishing methods as quantified by qPCR (single observations, n = 3 assay replicates, means ± SD). (b) Silver-stained SDS-polyacrylamide gel of each of the final AAV preparations relative to the common starting material (pool). A total of 2 × 109 VG was loaded per lane. M, marker lane. Asterisks indicate VP1, VP2, and VP3. (c) Bioactivity of the purified AAV preparations depending on the polishing method used. GFP-positive cells were detected by flow cytometry 48 hr after transduction at increasing multiplicity of infection (MOI). Single observations, n = 4 assay replicates, means ± SD. Color image available online at www.liebertpub.com/hgtb
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f4: Comparative analysis of polishing methods for AAVs isolated from an iodixanol gradient. AAV target fractions obtained from two parallel iodixanol gradients loaded with the HEK-293 cell lysate from a total of fifty 15-cm plates were further purified by ultrafiltration (UF), size-exclusion chromatography (SEC), hollow-fiber tangential flow filtration (HF-TFF), or PEG-8000 precipitation. (a) AAV vector yield and loss during purification by the various polishing methods as quantified by qPCR (single observations, n = 3 assay replicates, means ± SD). (b) Silver-stained SDS-polyacrylamide gel of each of the final AAV preparations relative to the common starting material (pool). A total of 2 × 109 VG was loaded per lane. M, marker lane. Asterisks indicate VP1, VP2, and VP3. (c) Bioactivity of the purified AAV preparations depending on the polishing method used. GFP-positive cells were detected by flow cytometry 48 hr after transduction at increasing multiplicity of infection (MOI). Single observations, n = 4 assay replicates, means ± SD. Color image available online at www.liebertpub.com/hgtb

Mentions: To assess the loss of AAV vectors associated with each of the purification methods, we first conducted qPCR quantification of the final AAV vector preparations relative to the common starting material. The results showed that ultrafiltration reached the highest AAV recovery at 92.6%, followed by SEC at 72.5%, HF-TFF at 70.3%, and PEG precipitation at 56.0% (Fig. 4a). SDS-PAGE analysis revealed that the AAVs purified by ultrafiltration, HF-TFF, and PEG precipitation were similarly clean as opposed to SEC-purified vectors, which showed a clearly higher amount of protein impurities (Fig. 4b). Moreover, whereas the concentration of residual iodixanol was <0.5% in AAV preparations purified by ultrafiltration and PEG precipitation, it was ∼3% in HF-TFF-purified samples and >5% in SEC-purified samples, as determined by mass spectrometry. Finally, in vitro bioactivity was assessed by transducing HEK-293 cells, which showed the slightly higher bioactivity of SEC-purified vectors as compared with cells transduced with vectors purified by the other polishing methods, which behaved similarly in this assay (Fig. 4c). Therefore, regarding the required time and efficiency to deplete iodixanol and contaminating proteins, ultrafiltration turned out to be the most efficient method for the final purification of iodixanol-derived AAV vectors.


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)

Comparative analysis of polishing methods for AAVs isolated from an iodixanol gradient. AAV target fractions obtained from two parallel iodixanol gradients loaded with the HEK-293 cell lysate from a total of fifty 15-cm plates were further purified by ultrafiltration (UF), size-exclusion chromatography (SEC), hollow-fiber tangential flow filtration (HF-TFF), or PEG-8000 precipitation. (a) AAV vector yield and loss during purification by the various polishing methods as quantified by qPCR (single observations, n = 3 assay replicates, means ± SD). (b) Silver-stained SDS-polyacrylamide gel of each of the final AAV preparations relative to the common starting material (pool). A total of 2 × 109 VG was loaded per lane. M, marker lane. Asterisks indicate VP1, VP2, and VP3. (c) Bioactivity of the purified AAV preparations depending on the polishing method used. GFP-positive cells were detected by flow cytometry 48 hr after transduction at increasing multiplicity of infection (MOI). Single observations, n = 4 assay replicates, means ± SD. 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

f4: Comparative analysis of polishing methods for AAVs isolated from an iodixanol gradient. AAV target fractions obtained from two parallel iodixanol gradients loaded with the HEK-293 cell lysate from a total of fifty 15-cm plates were further purified by ultrafiltration (UF), size-exclusion chromatography (SEC), hollow-fiber tangential flow filtration (HF-TFF), or PEG-8000 precipitation. (a) AAV vector yield and loss during purification by the various polishing methods as quantified by qPCR (single observations, n = 3 assay replicates, means ± SD). (b) Silver-stained SDS-polyacrylamide gel of each of the final AAV preparations relative to the common starting material (pool). A total of 2 × 109 VG was loaded per lane. M, marker lane. Asterisks indicate VP1, VP2, and VP3. (c) Bioactivity of the purified AAV preparations depending on the polishing method used. GFP-positive cells were detected by flow cytometry 48 hr after transduction at increasing multiplicity of infection (MOI). Single observations, n = 4 assay replicates, means ± SD. Color image available online at www.liebertpub.com/hgtb
Mentions: To assess the loss of AAV vectors associated with each of the purification methods, we first conducted qPCR quantification of the final AAV vector preparations relative to the common starting material. The results showed that ultrafiltration reached the highest AAV recovery at 92.6%, followed by SEC at 72.5%, HF-TFF at 70.3%, and PEG precipitation at 56.0% (Fig. 4a). SDS-PAGE analysis revealed that the AAVs purified by ultrafiltration, HF-TFF, and PEG precipitation were similarly clean as opposed to SEC-purified vectors, which showed a clearly higher amount of protein impurities (Fig. 4b). Moreover, whereas the concentration of residual iodixanol was <0.5% in AAV preparations purified by ultrafiltration and PEG precipitation, it was ∼3% in HF-TFF-purified samples and >5% in SEC-purified samples, as determined by mass spectrometry. Finally, in vitro bioactivity was assessed by transducing HEK-293 cells, which showed the slightly higher bioactivity of SEC-purified vectors as compared with cells transduced with vectors purified by the other polishing methods, which behaved similarly in this assay (Fig. 4c). Therefore, regarding the required time and efficiency to deplete iodixanol and contaminating proteins, ultrafiltration turned out to be the most efficient method for the final purification of iodixanol-derived AAV vectors.

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