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Type of publication: Journal Article
Type of document: Full Paper

Year: 2013

Authors: Gerster, P; Kopecky, EM; Hammerschmidt, N; Klausberger, M; Krammer, F; Grabherr, R; Mersich, C; Urbas, L; Kramberger, P; Paril, T; Schreiner, M; Nöbauer, K; Razzazi-Fazeli, E; Jungbauer, A

Title: Purification of infective baculoviruses by monoliths.

Source: J Chromatogr A. 2013; 1290:36-45



Authors Vetmeduni Vienna:

Nöbauer Katharina
Razzazi-Fazeli Ebrahim

Vetmed Research Units
VetCore


Abstract:
A chromatographic process based on monoliths for purification of infective baculovirus without prior concentration step has been established. Baculovirus produced in Spodoptera frugiperda cells (Sf-9) were harvested by centrifugation, filtered through 0.8 μm filters and directly loaded onto radial 1 mL anion exchange monoliths with a channel size of 1.5-2.0 μm operated at a volumetric flow rate of one bed volume per minute. Optional an epoxy monolith was used as pre-column to reduce interfering compounds and substances influencing the capacity of anion exchange monoliths for baculovirus infectious virus could be eluted with a step gradient at salt concentrations of 440 mM NaCl. Recovery of infectious virus was highly influenced by composition and age of supernatant and ranged from 20 to >99% active baculovirus. Total protein content could be reduced to 1-8% and DNA content to 38-48% in main virus fraction. Infective virus could be 52-fold concentrated within 20.5h and simultaneously an 82-fold volume reduction was possible when loading 1150 mL (2.1×10(8) pfu/mL) onto 1 mL scale support.

Keywords Pubmed: Animals
Anions/chemistry
Baculoviridae/isolation & purification*
Blotting, Western
Cell Culture Techniques
Chromatography, Ion Exchange/instrumentation
Chromatography, Ion Exchange/methods*
Electrophoresis, Polyacrylamide Gel
Lipids
Particle Size
Reproducibility of Results
Sf9 Cells/virology*


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