Empty capsids in column-purified recombinant adenovirus preparations

Empty capsids from adenovirus, that is, virus particles lacking DNA, are we ll documented in the published literature. They can be separated from compl ete 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 conta ining the p53 tumor suppressor gene was produced from 293 cells grown on mi crocarriers and purified by passage through DEAE-Fractogel and gel-filtrati on chromatography. Further sequential purification of the column-purified v irus by CsCl and glycerol density gradient centrifugations yielded isolated complete virus and empty capsids. The empty capsids were essentially nonin fectious 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 (pV III) by mass spectrometric analysis. No pVIII was detected from the purifie d complete virus. Analysis by electron microscopy of the empty capsids show ed 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 concen tration (as estimated by either light scattering or hexon content). They we re then used as a standard to establish the empty capsid concentration of v arious recombinant adenovirus preparations. Preliminary research showed cha nges in empty capsid concentration with variations in the infection conditi ons. While virus purification on anion-exchange or gel-filtration chromatog raphy has little effect on empty capsid contamination, other chromatographi c steps can substantially reduce the final concentration of empty capsids i n column-purified adenovirus preparations.