SIZE DISTRIBUTION ANALYSIS OF RECOMBINANT ADENOVIRUS USING DISC CENTRIFUGATION

Citation
Ll. Bondoc et S. Fitzpatrick, SIZE DISTRIBUTION ANALYSIS OF RECOMBINANT ADENOVIRUS USING DISC CENTRIFUGATION, Journal of industrial microbiology & biotechnology, 20(6), 1998, pp. 317-322
Citations number
34
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biothechnology & Applied Migrobiology
Volume
20
Issue
6
Year of publication
1998
Pages
317 - 322
Database
ISI
SICI code
Abstract
Recombinant adenovirus is one of the primary vectors for human gene th erapy. However, the aggregation of unstable virus has been a recurring problem during the production of purified virus for human therapeutic s. To facilitate the development of a robust manufacturing process for recombinant adenovirus vectors, a convenient and reliable size distri bution analytical assay is necessary and we demonstrate here that disc centrifuge sedimentation is applicable to this purpose. Using the dis c centrifuge system and the line start method, the assay can provide p article size distribution of adenovirus samples within 30 min. The ass ay can detect virus concentrations down to 0.01% (w/v) or 3 x 10(11) p articles per ml. The apparent hydrodynamic diameter of recombinant ade novirus was determined to be about 0.063 mu m. Furthermore, the disc c entrifuge analysis was able to detect adenovirus dimers, trimers, and tetramers, consistent with a rigid sphere approximation for adenovirus , as well as a large aggregate of 0.35 mu m. The appearance of viral a ggregates is confirmed by increased light scattering based on A(320)/A (260) ratios. The technique could be useful for monitoring the kinetic s of aggregation for adenovirus and other DNA and RNA viruses in the s ubmicron region. Therefore, this novel assay provides a critical tool for purification development of viral vectors for meeting therapeutic and research needs.