Development and validation of a HPLC method for the quantification of baculovirus particles.
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 8.9 min at a NaCl concentration of 480 mM NaCl (in 20 mM Tris-HCl, pH 7.5). The total run time of the method was 25 min. The method resulted in a linear response from 1×10(8) to 5.0×10(10)viral particles (VP/ml). The detection limit was 3.0×10(7) and the quantification limit was 1×10(8)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.
Animal Cell Technology Group, Bioprocess Center, Biotechnology Research Institute, National Research Council Canada, 6100 Royalmount Avenue, Montreal, Quebec, Canada H4P 2R2.
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D000818:Animals; D016367:Baculoviridae / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D052160:Benzothiazoles; D015153:Blotting, Western; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002852:Chromatography, Ion Exchange; D004279:DNA, Viral / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D003959:Diamines; D004591:Electrophoresis, Polyacrylamide Gel; D016679:Genome, Viral; D016014:Linear Models; D009930:Organic Chemicals / Q000737:chemistry / Q000378:metabolism; D011804:Quinolines; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D018411:Spodoptera; D014771:Virion / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D014773:Virology / Q000379:methods*; D014776:Virus Cultivation
D052160:Benzothiazoles; D004279:DNA, Viral; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I