Process intensification for high yield production of influenza H1N1 Gag virus-like particles using an inducible HEK-293 stable cell line |
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| Affiliation: | 1. Department of Chemical Engineering, École Polytechnique de Montréal, Montréal, Québec, Canada;2. Vaccine Program, Human Health Therapeutics, National Research Council Canada, Montréal, Québec, Canada;3. Department of Bioengineering, McGill University, Montréal, Québec, Canada;1. Novavax, Inc., 22 Firstfield, Gaithersburg, MD 20878, USA;2. Medigen, Inc., 8420 Gas House Pike, Frederick, MD, USA;3. Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA;4. Microbiology and Molecular Genetics Graduate Program, Emory University, Atlanta, GA, USA;1. Biotechnology Core Laboratory NIDDK, NIH, Bethesda, Maryland, 20892, USA;2. Department of Chemical and Biomolecular Engineering Johns Hopkins University, Baltimore, Maryland, 21218, USA;1. Karlsruhe Institute of Technology, Institute of Process Engineering in Life Sciences, Section IV: Biomolecular Separation Engineering, Karlsruhe, Germany;2. Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany;3. Diarect AG, Freiburg, Germany;1. Department of Chemical Engineering, Michigan Technological University, Houghton, MI 49931, USA;2. Health Research Institute, Michigan Technological University, Houghton, MI 49931, USA;3. Esperovax, Plymouth, MI 48170, USA;4. Department of Mechanical Engineering – Engineering Mechanics, Michigan Technological University, Houghton, MI 49931, USA;1. IBET – Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, Portugal;2. Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Avenida República, 2780-157 Oeiras, Portugal;3. Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, USA;1. Novavax, Inc., 20 Firstfield, Gaithersburg, MD 20878, USA;2. Medigen, Inc., 8420 Gas House Pike, Frederick, MD, USA;3. Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA;4. Microbiology and Molecular Genetics Graduate Program, Emory University, Atlanta, GA, USA |
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| Abstract: | Influenza virus dominant antigens presentation using virus like particle (VLP) approach is attractive for the development of new generation of influenza vaccines. Mammalian cell platform offers many advantages for VLP production. However, limited attention has been paid to the processing of mammalian cell produced VLPs. Better understanding of the production system could contribute to increasing the yields and making large-scale VLP vaccine manufacturing feasible. In a previous study, we have generated a human embryonic kidney HEK-293 inducible cell line expressing Hemagglutinin (HA) and Neuraminidase (NA), which was used to produce VLPs upon transient transfection with a plasmid containing HIV-1 Gag. In this work, to streamline the production process, we have developed a new HEK-293 inducible cell line adapted to suspension growth expressing the three proteins HA, NA (H1N1 A/PR/8/1934) and the Gag fused to GFP for monitoring the VLP production. The process was optimized to reach higher volumetric yield of VLPs by increasing the cell density at the time of induction without sacrificing the cell specific productivity. A 5-fold improvement was achieved by doing media evaluation at small scale. Furthermore, a 3-L perfusion bioreactor mirrored the performance of small-scale shake flask cultures with sequential medium replacement. The cell density was increased to 14 × 106 cells/ml at the time of induction which augmented by 60-fold the volumetric yield to 1.54 × 1010 Gag-GFP fluorescent events/ml, as measured by flow cytometry. The 9.5-L harvest from the perfusion bioreactor was concentrated by tangential flow filtration at low shear rate. The electron micrographs revealed the presence of VLPs of 100–150 nm with the characteristic dense core of HIV-1 particles. The developed process shows the feasibility of producing high quantity of influenza VLPs from an inducible mammalian stable cell line aiming at large scale vaccine manufacturing. |
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| Keywords: | Stable cell line Process development Perfusion TFF Gag-VLPs Influenza vaccine |
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