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

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 1 h. 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.Crown

Particle quantification of influenza viruses by high performance liquid chromatography

“…Ion exchange (IEX)-HPLC has been developed for the particle quantification of viral vectors for gene therapy and cancer therapy treatments produced in mammalian cell culture [17][18][19][20][21][22]. The use of this method in our group as an in-process monitoring tool was proven to be invaluable in the successful scale up of viral products that reached commercialization (ONRAB by Artemis Technologies, Canada) or are currently in late stage of clinical evaluation (Reolysin by Oncolytic Biotech., Canada).…”

Purification of a recombinant baculovirus of Autographa californica M nucleopolyhedrovirus by ion exchange membrane chromatography

“…This includes the use of ultrafiltration membranes, ion exchange membranes and resin-based chromatography (Barsoum, 1999;Michalsky et al, 2009;Transfiguracion et al, 2011;Wu et al, 2007). However, the purification of AcMPNV is developed poorly (Vicente et al, 2010;Vicente et al, 2009) and materials and processes for the efficient downstream processing of baculoviruses for clinical applications is sought (Vicente et al, 2010;Vicente et al, 2009).…”

Integrated Application of Bioprocess Engineering and Biotechniques for Quality & Bulk Drug Manufacturing

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“…The aspect ratio (H/D) of stirred vessels varies over a wide range. When aeration is required, the aspect ratio is usually increased [11,12,14]. This provides for longer contact times between the rising bubbles and liquid and produces a greater hydrostatic pressure at the bottom of the vessel.…”