An efficient process for the purification of helper-dependent adenoviral vector and removal of helper virus by iodixanol ultracentrifugation.
The preparation of large amount of purified helper-dependent adenoviral vector material is hampered by the lack of development of downstream processes with proven records on separation and recovery efficiencies. In order to facilitate the use of clinical-grade helper-dependent virus material for large-scale in vivo studies, a three-step purification scheme consisting of (1) an anion-exchange chromatography for initial capturing of virus, (2) a shallow iodixanol density gradient ultracentrifugation for the removal of helper virus from helper-dependent virus, and (3) a size-exclusion chromatography for the removal of iodixanol and residual protein contaminants as a polishing step was developed. The use of a fast iodixanol density ultracentrifugation step was highly effective in separating infectious helper-dependent virus from contaminating helper virus. The overall downstream processing scheme gave 80% infectious particle yield. The contamination ratio of helper virus in the helper-dependent virus preparation are reduced from 2.57 to 0.03% corresponding to a reduction of helper virus by factors of 85 by two iodixanol purification steps. It was also demonstrated that size-exclusion chromatography is an excellent step for the removal of iodixanol and polishing of the final helper-dependent virus preparation.
Dormond
Edwige
E
Animal Cell Technology Group, Biotechnology Research Institute, National Research Council Canada, 6100 Royalmount Avenue, Montréal, Québec, Canada.
Chahal
Parminder
P
Bernier
Alice
A
Tran
Rosa
R
Perrier
Michel
M
Kamen
Amine
A
D000256:Adenoviridae / Q000302:isolation & purification*; D002850:Chromatography, Gel / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D005822:Genetic Vectors / Q000302:isolation & purification*; D006378:Helper Viruses / Q000302:isolation & purification*; D006801:Humans; D014283:Triiodobenzoic Acids; D014461:Ultracentrifugation / Q000379:methods*; D014773:Virology / Q000379:methods*
D014283:Triiodobenzoic Acids; C044834:iodixanol