Adenoviral, AAV, and retroviral vectors are produced in mammalian cells. One way to release Ad and AAV vectors from cell pellets is by applying multiple freeze-thaw cycles. Retroviruses such as LV on the other hand are released into the supernatant. The viral vectors may be separated from the cellular debris by either centrifugation followed by filtration or by using a series of filters with decreasing porosity.
A classical method of Ad vector purification has involved cesium chloride (CsCl) density gradient centrifugation. This process typically takes one to two days and generates vector stocks of variable quality. A major drawback of this method is its limited scalability making it unsuitable for large-scale vector production.
Although Ad have been traditionally isolated by CsCl density gradient ultracentrifugation, recently other purification methods based on ion exchange chromatography [17-19], size exclusion chromatography [20,21] and hydrophobic interaction chromatography  have been reported. A general Ad purification scheme shown in Figure 20.1 summarizes some of the key steps. Goerke and coworkers  have recently reported an Ad purification process that involves selective precipitation of host cell DNA as well as proteins with 2% domiphen bromide, a cationic detergent resulting in 3 log reduction of DNA with host cell protein levels of 15 ^g/1011 viral particles in yields of 58 to 86% as determined by anion exchange chromatography. Examples in published literature however, report either one- or two-step chromatography processes followed by ultrafiltration, diafiltration, and sterile filtration. As the
Ad capsid protein composition changes with the serotype, the ionic charge on the Ad particle can be modulated in solution. Ion exchange chromatography can therefore be used for the capture step.
Huyghe and coworkers  reported on a two-column process to purify Ad vectors. Approximately 3 x 1012 Ad particles present in a crude cell lys-ate were captured on a 1.7 ml DEAE anion exchange column at a flow rate of 1 ml/min. A follow-up step included a zinc metal ion affinity column. The overall recovery was 32% and the purity of the virus preparation was good, as judged by protein gel analysis. Green and coworkers  purified Ad vectors using DEAE anion exchange column chromatography and reversed phase ion-pair chromatography. Kamen and Henry  as well as Arcand and coworkers  have developed a process that involved capture of Ad particles from 20 l of lysate on a DEAE anion exchange column resulting in 80% recovery. The first chromatography step was followed by a polishing step using gel filtration.
Cesium chloride density gradient centrifugation has been used successfully in the past for the purification of AAV-2 vectors resulting in virus particles of sufficient purity to generate x-ray diffraction quality crystals . However, this approach is limited due to the poor scalability in the production of sufficient vector quantities to meet the demands of Phase III clinical trials and beyond.
Purification of AAV-5 using mucin affinity chromatography involving agarose beads has been reported to result in vector preparations whose purity was comparable to that of vectors purified by CsCl gradient centrifugation . Several groups have used heparin affinity chromatography for the purification of AAV-2 and -3 vector stocks . Zolotukhin et al.  purified AAV vectors by centrifugation using discontinuous iodixanol gradients followed by heparin affinity chromatography resulting in 30 to 60% overall recovery. Snyder and Flotte  described a three-step purification procedure involving capture of the vector on a heparin column followed by a purification step using phenyl hydrophobic interaction chromatography, and a final polishing step involving another heparin column. While affinity chromatography has been demonstrated in the literature as a good alternative for the purification of AAV vectors, it has several limitations. Some of these include the fact that affinity ligands for some of the AAV serotypes such as type 8 have not been identified. Also, the possibility of affinity ligands leaching into the purified product further complicates the usefulness of this method to generate clinical-grade AAV vector stocks.
As with Ad vectors, AAV vectors also display different capsid protein compositions depending on the serotype. Therefore, one can utilize ion exchange chromatography to exploit the differences in the ionic charge properties of these vectors in solution. Several AAV vector purification methods involving beaded ion exchange chromatography media have been reported. These included (a) a vector capture step using a heparin column followed by another purification step involving a PEI anion exchange column ; (b) a capture and purification step using either a PEI or Q anion exchange chromatography column ; (c) a two-step purification protocol involving a strong cation exchange chroma-tography resin followed by a strong anion exchange resin ; and (d) capture of the AAV vector by anion exchange chromatography using a strong anion exchange resin with subsequent polishing by gel filtration chromatography .
Lentiviral vector production for large-scale in vivo applications that require high-titer stocks is challenging due to the lack of simple procedures capable of rapidly processing large volumes of cell culture supernatant. The traditional ultracentrifugation-based approaches are limited in terms of their capacity to handle large volumes, thus making this procedure extremely tedious. One problem with ultracentrifugation-based approaches is that cell-derived components are concentrated along with the vector particles leading to potential immune and inflammation responses .
Thus, chromatography-based approaches are needed in order to purify lentivirus vectors of contaminating host cell components. Methods based on anion exchange chromatography of HIV-1 vectors pseudotyped with VSV-G have been established [36,37]. Schauber et al.  described a similar procedure for HIV-1 vectors pseudotyped with the baculovirus GP64 glycoprotein. Yields and purity of the virus stocks resulting from these procedures were not reported, but these approaches may lead to vector stocks of improved purity, increased infectivity, and reduced toxicity. The purification of inactivated HIV-1 particles that involved a two-step TMAE and Q anion exchange chromatography procedure yielding virus preparations with >95% purity as judged by gel filtration chromatography analysis was also reported . Size-exclusion chromatography has also been used to purify HIV-1 vectors albeit with a prior concentration of the cell culture supernatant by cross-flow filtration [3,40].
Plasmid DNA is typically produced in bacterial cells such as Escherichia coli. Plasmid DNA can be released from bacterial cell pellets by alkaline cell lysis . The lysate containing plasmid DNA is neutralized using potassium acetate resulting in the precipitation of proteins along with the cellular debris including bacterial DNA . Addition of high concentrations of calcium chloride to the lysate prior to clarification has been reported to effectively precipitate most of the RNA contaminants . Clarification of the plasmid DNA containing lysate can be achieved by centrifugation or by dead-end filtration consisting of two filters in series with decreasing porosity . Plasmid DNAs from clarified cell lysates are then purified further by different techniques such as CsCl gradient ultracentrifugation in the presence of ethidium bromide, or ion exchange chromatography using beaded, or membrane media. Plasmid DNA purification methods involving anion exchange chromatography has been reported by Schleef  and by Prazeres and coworkers . The former used 15% isopropanol in the wash and elution buffers. The high costs of the buffer and handling and disposal expenses could make such a process economically unfavorable during scale-up. A general purification scheme based on literature examples for plasmid DNA is summarized in Figure 20.2.
In most instances, a reduction in the RNA impurities is generally carried out prior to the plasmid capture by anion exchange chromatography. But this requires an additional diafiltration step before the chromatography step. Several modifications of the process shown in Figure 20.2 that are discussed below have
been explored recently in an attempt to address critical plasmid purification process issues such as plasmid capacity on chromatography media, plasmid purity with respect to RNA, and endotoxin contamination as well as plasmid recovery.
Levy and coworkers  used a similar but more elaborate plasmid purification process that involved RNase A digestion, partial purification of plasmid DNA by PEG precipitation, followed by filtration involving nitrocellulose filters before capture on anion exchange chromatography media, with further purification by another nitrocellulose filtration step and an anion exchange chromatography column. Eon-Duval and Burke  screened several anion exchange chromatography sorbents for plasmid polishing for trace RNA removal following primary purification using precipitation and tangential flow filtration (TFF). They found that for an approximately 5 kb plasmid, polishing on both Fractogel®* DEAE and POROS®** 50HQ resulted in >98.0% RNA
* Fractogel is a registered trademark of Merck KGaA Darmstadt.
"POROS is a registered trademark of Applied Biosystems.
removal with > 94.0% plasmid recovery when the plasmid was loaded in 0.63 M NaCl and 0.72 M NaCl, respectively, in 50 mM phosphate buffer pH 7.0 at a flow rate of 150 cm/h.
Hydroxyapatite chromatography media have been shown to be useful for plasmid DNA purification . Giovannini and Freitag  reported on the effects of the ratio of calcium and phosphorous in such media on the dynamic binding capacities of plasmid DNA. They concluded that the Hydroxyapatite media containing low calcium to phosphorous (C/P) ratios resulted in the best binding capacities (446 \xg/g for a 4.7 kb plasmid and 59 \xg for 11.4 kb plasmid). Sagar and coworkers  have shown that the dynamic binding capacity of some reversed phase beaded chromatography media can be increased by two- to threefold by adding up to 1 M NaCl to a plasmid feed stream.
Alternative protocols for the purification of plasmid DNA were also reported. Horn and coworkers  reported an overall yield of 50% following two successive PEG precipitations followed by a size-exclusion chromatography step. Lander and coworkers  demonstrated that cetyltrimethylammonium bromide (CTAB) selectively precipitated plasmid DNA from proteins, genomic DNA, RNA, and endotoxin. TFF has been shown to be effective in removing >99% of the contaminating RNAs from plasmid DNA after a precipitation step involving high concentrations of calcium chloride followed by centrifu-gation and micro-filtration . Alternatively, by extending the alkaline lysis step in 0.2 M NaOH and 1% SDS from under 30 min to 24 h resulted in lower levels of RNA and endotoxin in a lysate containing a 10 kb plasmid while further reduction of these contaminants was accomplished by TTF . Prazeres et al.  have recently published a detailed review of plasmid DNA purification by different chromatography techniques.
Thus, several methods exist for capture, purification, and polishing of gene therapy vectors that include both chromatographic as well as nonchromato-graphic techniques. All of the methods discussed above have their merits and drawbacks that impact the decision to adopt them for process development and ultimately transfer to manufacturing scale. The following sections will discuss membrane chromatography purification for capture of Ad, capture, and purification of AAV and for capture of LV.
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