Empty Capsids in Column-Purified Recombinant Adenovirus Preparations

Abstract

Empty capsids from adenovirus, that is, virus particles lacking DNA, are well documented in the published literature. They can be separated from complete virus by CsCl density gradient centrifugation. Here we characterize the presence of empty capsids in recombinant adenovirus preparations purified by column chromatography. The initial purified recombinant adenovirus containing the p53 tumor suppressor gene was produced from 293 cells grown on microcarriers and purified by passage through DEAE-Fractogel and gel-filtration chromatography. Further sequential purification of the column-purified virus by CsCl and glycerol density gradient centrifugations yielded isolated complete virus and empty capsids. The empty capsids were essentially noninfectious and free of DNA. Analysis of empty capsids by SDS-PAGE or RP-HPLC showed the presence of only three major components: hexon, IIIa, and a 31K band. This last protein was identified as the precursor to protein VIII (pVIII) by mass spectrometric analysis. No pVIII was detected from the purified complete virus. Analysis by electron microscopy of the empty capsids showed particles with small defects. The amount of pVIII was used to determine the level of empty capsid contamination. First, the purified empty capsids were used to quantify the relation of pVIII to empty capsid particle concentration (as estimated by either light scattering or hexon content). They were then used as a standard to establish the empty capsid concentration of various recombinant adenovirus preparations. Preliminary research showed changes in empty capsid concentration with variations in the infection conditions. While virus purification on anion-exchange or gel-filtration chromatography has little effect on empty capsid contamination, other chromatographic steps can substantially reduce the final concentration of empty capsids in column-purified adenovirus preparations.

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

“…Analytical HPLC is one of the most useful techniques for analysis of both pure and crude samples of Ad 17,[66][67][68][69] and AAV. 70 HPLC using anion-exchange column Source 15 Q (Amersham Biosciences) for analysis of Ad particles in crude lysates 66 offers a significant advantage over the semiquantitative and time-consuming assays available previously.…”

“…While some chromatographic steps for Ad purification, such as DEAE-Fractogel chromatography, are not suitable for capsid elimination, other chromatographic methods, including immobilized zincchelating or hydrophobic interaction chromatography, can substantially reduce contamination with empty viruses. 69 Purification of rAAV particles by ion-exchange chromatography cannot discriminate between full and empty AAV capsids. 49,54 A method for quantification of Ad capsids by RP-HPLC is based on measuring the amounts of capsid protein VIII precursor.…”

“…This protein is present in empty capsids but not in intact Ad particles. 69 Perspectives for the purification of adenoviral and adeno-associated viral vectors…”

Removal of envelope protein‐free retroviral vectors by anion‐exchange chromatography to improve product quality

“…It is also possible that the lipid bilayer of the viral particle increases the strength of adsorption of the Env + vectors to the AEXc matrix by locally affecting the electrostatic properties of the envelope proteins at pH values higher than the pI of Env -vectors [37]. These effects are not observed in nonenveloped viral particles (e. g., adenoviruses and rAAV), where the protein capsids adsorbed to the chromatographic matrix are displaced by NaCl concentrations lower than 600 mM [38], compared with 600 -1000 mM NaCl required for RVs. In addition, significantly higher fpotential values ( -8.6 mV at pH 7.3 for rAAV [39]), thus lower surface charge density, have been reported for nonenveloped viruses [39].…”

Purification of adenovirus and adeno-associated virus: comparison of novel membrane-based technology to conventional techniques

“…Further improved high performance liquid chromatography (HPLC) methods have been described [4][5][6] coupled with the use of DEAE-Fractogel and zinc affinity chromatography. 7 Currently, there are commercially available kits, which use membrane-based technology as an alternative to double CsCl centrifugation. Adenopuret is based on membrane absorber technology, which allows the binding of ion exchange functional groups to the inner surface of synthetic microporous membranes in a syringe-filter format.…”