An improved anion-exchange HPLC method for the detection and purification of adenoviral particles

Blanche, Francis; Cameron, Béatrice; Barbot, Anne; Ferrero, Lucy; Guillemin, Thierry; Guyot, Sylvain; Somarriba, S; Bisch, D

doi:10.1038/sj.gt.3301190

Cited by 86 publications

(75 citation statements)

References 24 publications

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“…In addition to the conventional 2× CsCl method, a number of column-based procedures have been reported for the purification of adenovectors, including ion-exchange [27-31], metal chelation [30], size-exclusion [27,[29][30][31], and affinity [28,30] chromatography. Although these column-based methods may improve scalability, recovery rate, and sometimes purity of production, they are usually complicated and require multiple steps with more contingent and harsh elution conditions [27,28,30,31] that can be time-consuming and costly.…”

Section: Discussionmentioning

confidence: 99%

“…Although these column-based methods may improve scalability, recovery rate, and sometimes purity of production, they are usually complicated and require multiple steps with more contingent and harsh elution conditions [27,28,30,31] that can be time-consuming and costly. Conventional 2× CsCl gradient centrifugation has therefore remained a common method for mid-scale production of adenovirus in academic laboratories.…”

Section: Discussionmentioning

confidence: 99%

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A rapid and efficient method for purification of recombinant adenovirus with arginine–glycine–aspartic acid-modified fibers

Peng¹,

Wu²,

Davis³

et al. 2006

Analytical Biochemistry

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Recombinant adenoviral vectors (adenovectors) have been subject to various genetic modifications to improve their transduction efficiency and targeting capacity. Production and purification of adenovectors with modified capsid proteins can be problematic using conventional two-cycle CsCl gradient ultracentrifugation. We have developed a new method for purifying recombinant adenovectors in two steps: iodixanol discontinuous density gradient ultracentrifugation and sizeexclusion column chromatography. The purity and infectious activity of adenovectors isolated by either method were comparable. The new method yielded three to four times more adenovectors with RGD-modified fiber proteins than did the conventional CsCl method. For other fiber-modified and wild-type adenovectors, the yields of the two methods were comparable. Thus, the iodixanol-based method can be used not only to improve the production of RGD-modified adenovectors, but also to purify adenovectors with or without fiber modifications. Moreover, the whole procedure can be completed in 3 hours. Therefore, this method is rapid and efficient for production recombination adenovectors, especially those with RGD-modified fibers.Adenoviral vectors (adenovectors) have high in vivo transduction efficiencies and are easy to construct and produce. They are thus frequently used for in vitro and in vivo gene delivery and for gene therapy in clinical trials [1] . Substantial efforts have been made to characterize adenovirus-host cell interactions and to improve gene delivery by adenovectors, including minimizing vector gene expression, reducing vector related toxicity and immunogenicity, increasing their capacity to accommodate large foreign genes, and increasing their transduction efficiency.It is now known that adenovirus interacts with host cells by binding to the coxsackievirus and adenovirus receptor (CAR) [2], a cellular receptor for Ad5 and most other adenovirus serotypes. After attachment, the virion is then internalized by endocytosis through the interaction of its penton base with α v β 3 and α v β 5 integrins on the host cell surface [3]. However, there is growing evidence that the expression of CAR is frequently suppressed in various types of cells, including tumor cells and in primary tumors [4,5], resulting in resistance to adenovirus infection [6][7][8] Unfortunately, technical difficulties may be encountered in production of certain modified adenovectors. We have encountered difficulties when producing certain adenovectors by this method, especially those with RGD-modified fibers or "sticky" virus. For example, we have experienced difficulty in purifying various RGD-modified adenoviral vectors by conventional two-cycle (2x) of CsCl ultracentrifugation because the RGD-modified adenoviral vectors tend to aggregate, especially in the second round of ultracentrifugation, forming floccules without a sharp band. This results in a low yield or even failure of purification. Thus, an alternative method for efficient production of these modified adenov...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

A rapid and efficient method for purification of recombinant adenovirus with arginine–glycine–aspartic acid-modified fibers

Peng¹,

Wu²,

Davis³

et al. 2006

Analytical Biochemistry

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“…Hexon protein (II) may mediate the strongest interaction, since it has a net negative charge of 24.8 at pH 7.0 and it is highly abundant in the particle. A trend was found between the charge of the hexon L1 loop of different Ad serotypes and their binding to anion exchanger, 17 indicating that hexon L1 loop is very likely to be a major determinant of the strong interaction of the Ad particle with the adsorbent. Owing to its strong negative charge, Ad particle binds to an anion-exchange resin at high salt concentration, whereas most proteins elute from the column under the same conditions, allowing for an efficient separation from proteins.…”

Section: Purification By Ion-exchange Chromatographymentioning

confidence: 97%

“…The ion-exchange-based approach takes advantage of retention of Ad particles, which have an acidic pI at physiological conditions, on an anionexchange resin. [17][18][19] Efficient binding of Ad particles to charged divalent zinc ions has been exploited for developing Ad purification methods using immobilized metal affinity chromatography (IMAC). Importantly, the large size of Ad particles results in physical trapping of the virus in a column resin, becoming the source of significant viral loss during purification by some chromatographic methods.…”

Section: Adenoviruses and Adeno-associated Viruses As Vectors For In mentioning

confidence: 99%

“…As such, a number of anion-exchange adsorbents have been used successfully for the chromatographic purification of Ad. [17][18][19] The resolution of Ad structure by X-ray crystallography 53 indicates that functional groups of the proteins II, III, IIIa, IV and IX mediate the interaction between viral particles and the anion exchanger. Hexon protein (II) may mediate the strongest interaction, since it has a net negative charge of 24.8 at pH 7.0 and it is highly abundant in the particle.…”

Section: Purification By Ion-exchange Chromatographymentioning

confidence: 99%

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Chromatographic purification of recombinant adenoviral and adeno-associated viral vectors: methods and implications

Burova

Ioffe

2005

Gene Ther

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Anion exchange chromatography provides a robust, predictable process to ensure viral safety of biotechnology products

Strauss

Gorrell

Plancarte

et al. 2008

Biotech & Bioengineering

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The mammalian cell-lines used to produce biopharmaceutical products are known to produce endogenous retrovirus-like particles and have the potential to foster adventitious viruses as well. To ensure product safety and regulatory compliance, recovery processes must be capable of removing or inactivating any viral impurities or contaminants which may be present. Anion exchange chromatography (AEX) is a common process in the recovery of monoclonal antibody products and has been shown to be effective for viral removal. To further characterize the robustness of viral clearance by AEX with respect to process variations, we have investigated the ability of an AEX process to remove three model viruses using various combinations of mAb products, feedstock conductivities and compositions, equilibration buffers, and pooling criteria. Our data indicate that AEX provides complete or near-complete removal of all three model viruses over a wide range of process conditions, including those typically used in manufacturing processes. Furthermore, this process provides effective viral clearance for different mAb products, using a variety of feedstocks, equilibration buffers, and different pooling criteria. Viral clearance is observed to decrease when feedstocks with sufficiently high conductivities are used, and the limit at which the decrease occurs is dependent on the salt composition of the feedstock. These data illustrate the robust nature of the AEX recovery process for removal of viruses, and they indicate that proper design of AEX processes can ensure viral safety of mAb products.

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An improved anion-exchange HPLC method for the detection and purification of adenoviral particles

Cited by 86 publications

References 24 publications

A rapid and efficient method for purification of recombinant adenovirus with arginine–glycine–aspartic acid-modified fibers

A rapid and efficient method for purification of recombinant adenovirus with arginine–glycine–aspartic acid-modified fibers

Chromatographic purification of recombinant adenoviral and adeno-associated viral vectors: methods and implications

Anion exchange chromatography provides a robust, predictable process to ensure viral safety of biotechnology products

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