title: Rapid and reliable quantification of reovirus type 3 by high performance liquid chromatography during manufacturing of Reolysin.
abstract: Reolysin, a human reovirus type 3, is being evaluated in the clinic as an oncolytic therapy for various types of cancer. To facilitate the optimization and scale-up of the current process, a high performance liquid chromatography (HPLC) method has been developed that is rapid, specific and reliable for the quantification of reovirus type 3 particles. Using an anion-exchange column, the intact virus eluted from the contaminants in 9.78 min at 350 mM NaCl in 50mM HEPES, pH 7.10 in a total analysis time of 25 min. The virus demonstrated a homogenous peak with no co-elution of other compounds as analyzed by photodiode array analysis. The HPLC method facilitated the optimization of the purification process which resulted in the improvement of both total and infectious particle recovery and contributed to the successful scale-up of the process at the 20 L, 40 L and 100 L production scale. The method is suitable for the analysis of crude virus supernatants, crude lysates, semi-purified and purified preparations and therefore is an ideal monitoring tool during process development and scale-up.
authors.affiliation: Animal Cell Technology Group, Bioprocess Sector, Biotechnology Research Institute, National Research Council Canada, 6100 Royalmount Avenue, Montreal, Quebec, Canada H4P 2R2.
authors.forename: Julia
authors.initials: J
authors.lastname: Transfiguracion
authors.forename: Alice
authors.initials: A
authors.lastname: Bernier
authors.forename: Robert
authors.initials: R
authors.lastname: Voyer
authors.forename: Helene
authors.initials: H
authors.lastname: Coelho
authors.forename: Matt
authors.initials: M
authors.lastname: Coffey
authors.forename: Amine
authors.initials: A
authors.lastname: Kamen
mesh_terms: D002460:Cell Line; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D007668:Kidney / Q000166:cytology; D012089:Mammalian orthoreovirus 3 / Q000254:growth & development* / Q000302:isolation & purification* / Q000648:ultrastructure; D010316:Particle Size; D012680:Sensitivity and Specificity; D013997:Time Factors