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

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DOIResolve DOI: http://doi.org/10.1016/j.jchromb.2010.11.011
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TypeArticle
Journal titleJournal of Chromatography B
Volume879
Issue1
Pages6168; # of pages: 8
SubjectBaculovirus; HPLC; Quantification; Total virus particle; Viral genome labeling
AbstractA 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+ù10<sup>8</sup> to 5.0+ù10<sup>10</sup>viral particles (VP/ml). The detection limit was 3.0+ù10<sup>7</sup> and the quantification limit was 1+ù10<sup>8</sup>VP/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.
Publication date
LanguageEnglish
AffiliationNRC Biotechnology Research Institute; National Research Council Canada
Peer reviewedYes
NRC number52769
NPARC number16616684
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Record identifierb0bd7562-494c-4518-bcd4-ef48f0593528
Record created2011-03-31
Record modified2016-05-09
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