Particle quantification of influenza viruses by high performance liquid chromatography |
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| Institution: | 1. National Research Council Canada, Human Health Therapeutics, Montreal, Quebec, Canada;2. Department of Bioengineering, McGill University, Montreal, Quebec, Canada;1. BioProcessing R&D, GE Healthcare Bio-Sciences AB, Björkgatan 30, SE-751 84 Uppsala, Sweden;2. Institute for Translational Vaccinology, Antonie van Leeuwenhoeklaan 9, Pb 450, 3720 AL Bilthoven, The Netherlands;1. Serum Institute of India Private Limited, 212/2, Hadapsar, Pune, India;2. PATH, PO Box 900922, Seattle, WA 98109, USA;1. National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China;2. University of Chinese Academy of Sciences, Beijing 100049, PR China;1. State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China;2. University of Chinese Academy of Sciences, Beijing 100049, PR China;3. Hualan Biological Engineering, Inc, Xinxiang 453003, PR China;4. Key Laboratory of Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, PR China;5. CAS Key Laboratory of Green Process and Engineering, State Key Laboratory of Multiphase Complex Systems, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China;1. BIA Separations, Mirce 21, SI-5270 Ajdov??ina, Slovenia;2. AVIR Green Hills Biotechnology AG, Forsthausgasse 11, A-1200 Vienna, Austria;1. Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, 39106 Magdeburg, Germany;2. Faculty of Pharmaceutical Biotechnology, Biberach University of Applied Sciences, 88400 Biberach, Germany;3. Department of Biotechnology, Bielefeld University of Applied Sciences, 33615 Bielefeld, Germany;4. Biotechnology, Brandenburg University of Technology Cottbus—Senftenberg, 01968 Senftenberg, Germany;5. Chair of Bioprocess Engineering, Otto-von-Guericke University, 39106 Magdeburg, Germany |
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| Abstract: | The influenza virus continuously undergoes antigenic evolution requiring manufacturing, validation and release of new seasonal vaccine lots to match new circulating strains. Although current production processes are well established for manufacturing seasonal inactivated influenza vaccines, significant limitations have been underlined in the case of pandemic outbreaks. The World Health Organization called for a global pandemic influenza vaccine action plan including the development of new technologies. A rapid and reliable method for the quantification of influenza total particles is crucially needed to support the development, improvement and validation of novel influenza vaccine manufacturing platforms. This work presents the development of an ion exchange-high performance liquid chromatography method for the quantification of influenza virus particles. The method was developed using sucrose cushion purified influenza viruses A and B produced in HEK 293 suspension cell cultures. The virus was eluted in 1.5 M NaCl salt with 20 mM Tris–HCl and 0.01% Zwittergent at pH 8.0. It was detected by native fluorescence and the total analysis time was 13.5 min. A linear response range was established between 1 × 109 and 1 × 1011 virus particle per ml (VP/ml) with a correlation coefficient greater than 0.99. The limit of detection was between 2.07 × 108 and 4.35 × 109 whereas the limit of quantification was between 6.90 × 108 and 1.45 × 1010 VP/ml, respectively. The coefficient of variation of the intra- and inter-day precision of the method was less than 5% and 10%. HPLC data compared well with results obtained by electron microscopy, HA assay and with a virus counter, and was used to monitor virus concentrations in the supernatant obtained directly from the cell culture production vessels. The HPLC influenza virus analytical method can potentially be suitable as an in-process monitoring tool to accelerate the development of processes for the manufacturing of influenza vaccines. |
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| Keywords: | Influenza HPLC Quantification Vaccine Particle Pandemic |
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