National Research Council of Canada. Human Health Therapeutics
2014 Monolith Event: Summer School Symposium, May 30-June 4 2014, Adjovščina, Potorož, Slovenia
A high performance liquid chromatography (HPLC) method using a strong anion exchange monolithic column based on convective interaction media) CIM® technology was developed for the particle quantification of influenza viruses. The virus which was specifically detected by native fluorescence eluted in 5.55 min at 1.5 M NaCl gradient in 20 mM Tris-HCl + o.01% Zwittergent, pH 8.0 in a total analysis time of 13.5 min. The linearity of a curve was demonstrated for all inluenza virus investigated with a good correlation coefficient (R2) greater than 0.995. Among all the virus investigated, the detection limit of the method ranged between 2.07x108 and 4.35x109 while the quantification limit ranged between 6.90x108 and 1.45x1010 virus particle per ml (VP/ml), respectively. The intra- and inter-assay precision of the method were less than 5% and 10% respectively. The method which was developed using sucrose cushion purified influenza viruses was shown to be suitable in the analysis of cell culture supernatants making it an ideal in-process monitoring tool to facilitate the development of influenza vaccine manufacturing processes.