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Development and validation of a HPLC method for the quantification of baculovirus particles

Authors
Journal
Journal of Chromatography B
1570-0232
Publisher
Elsevier
Publication Date
Volume
879
Issue
1
Identifiers
DOI: 10.1016/j.jchromb.2010.11.011
Keywords
  • Baculovirus
  • Hplc
  • Quantification
  • Total Virus Particle
  • Viral Genome Labeling
Disciplines
  • Biology

Abstract

Abstract A HPLC method using an anion exchange column was developed for the quantification of baculovirus particles. To properly detect the virus eluting from the column, a nucleic acid dye was used to amplify the signal projected by the virus. The viral genome was labeled by incubating the virus with SYBR Green I at 37°C for a minimum of 1h. The virus was specifically eluted from the contaminants in 8.9min at a NaCl concentration of 480mM NaCl (in 20mM Tris–HCl, pH 7.5). The total run time of the method was 25min. The method resulted in a linear response from 1×108 to 5.0×1010viral particles (VP/ml). The detection limit was 3.0×107 and the quantification limit was 1×108VP/ml. The intra-assay precision was <10% for both purified and crude virus preparations whereas the inter-assay precisions were <5% and <10% for purified and crude virus preparations, respectively. The recovery/accuracy of the method ranged from 78 to 101%. This method is a robust monitoring tool to facilitate research activities with baculovirus vector and accelerate development of baculovirus-based processes for manufacturing of biologics.

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