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Published on: May 6, 2015
Empty capsids in column-purified recombinant adenovirus preparations
G Vellekamp1, F W Porter, S Sutjipto
1Biotechnology Development Department, Schering-Plough Research Institute, Union, NJ 07083, USA. gary.vellekamp@spcorp.com
Human Gene Therapy
|October 9, 2001
Summary
This study characterizes empty adenovirus capsids in purified recombinant adenovirus preparations. The precursor to protein VIII (pVIII) was identified as a marker for empty capsid contamination, enabling quantification and reduction strategies.
Area of Science:
- Virology
- Biochemistry
- Biotechnology
Background:
- Empty capsids, virus particles lacking DNA, are known contaminants in adenovirus preparations.
- Separation of empty and complete adenovirus particles is typically achieved via CsCl density gradient centrifugation.
Purpose of the Study:
- To characterize empty capsids in recombinant adenovirus purified by column chromatography.
- To identify a reliable marker for quantifying empty capsid contamination.
- To investigate methods for reducing empty capsid levels in purified adenovirus preparations.
Main Methods:
- Recombinant adenovirus was purified using DEAE-Fractogel and gel-filtration chromatography, followed by CsCl and glycerol density gradient ultracentrifugation.
- Empty capsids were analyzed by SDS-PAGE, RP-HPLC, mass spectrometry, and electron microscopy.
- The precursor to protein VIII (pVIII) was identified as a specific component of empty capsids.
Main Results:
- Purified empty capsids were noninfectious and DNA-free, containing hexon, IIIa, and a 31K band identified as pVIII.
- pVIII was absent in purified complete virus particles.
- A method was established to quantify empty capsid concentration using pVIII as a marker.
- Certain chromatographic steps were found to effectively reduce empty capsid contamination.
Conclusions:
- The precursor to protein VIII (pVIII) serves as a reliable indicator for empty capsid contamination in recombinant adenovirus.
- Quantification of pVIII allows for accurate assessment of empty capsid levels.
- Optimized chromatographic purification strategies can significantly minimize empty capsid contamination in therapeutic adenovirus preparations.
