Production of adenoviral vectors and its recovery |
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| Institution: | 1. Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA 15261, USA;2. Geriatric Research Educational and Clinical Center, V.A. Pittsburgh Healthcare System, Pittsburgh, PA 15261, USA;3. Department of Neurology, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15261, USA;4. Division of Pediatric Emergency Medicine, Department of Pediatrics, Children''s Hospital of Pittsburgh, Pittsburgh, PA 15261, USA;5. Safar Center for Resuscitation Research, University of Pittsburgh, Pittsburgh, PA 15261, USA;1. Graduate Program in Cell, Molecular, and Structural Biology, Miami University, Oxford, OH, 45056 USA;2. Department of Microbiology, Miami University, Oxford, OH, 45056 USA;1. Center for Nanotechnology Innovation @NEST, Istituto Italiano di Tecnologia, Piazza San Silvestro 12, 56127 Pisa, Italy;2. NEST, Scuola Normale Superiore and Istituto Nanoscienze-CNR, Piazza San Silvestro 12, 56127, Pisa, Italy;3. Department of Molecular Medicine, “Sapienza” University of Rome, Viale Regina Elena 291, 00161, Rome, Italy;1. Department of Marine, Earth, and Atmospheric Sciences, North Carolina State University, Raleigh, NC 27606, USA;2. Institute of Atmospheric Physics, Chinese Academy of Sciences, Beijing, China;3. Department of Air Quality and Environmental Management, Clark County, NV 89155, USA;4. Toyota Motor Engineering & Manufacturing, North America, INC., Ann Arbor, MI 48105, USA |
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| Abstract: | The current demands for adenoviral vectors are increasing to satisfy pre-clinical and clinical gene therapy protocols. Consequently, there is a necessity of methodologies to improve production and recovery of intact particles with the minimum effect upon bioactivity. The production of adenoviral vectors in HEK 293 cells and the potential of an alternative aqueous two-phase system (ATPS) composed of PEG 300-phosphate in recovery of adenoviral vectors were investigated. The production of adenoviral vectors was carried out using a 2 L bioreactor equipped with two Rushton impellers. Different parameters including initial cell density, harvesting time and the addition of a buffer (HEPES) were studied in order to improve the production of adenoviral vectors in HEK 293 cells. A yield of 8 × 1011 infective particles was achieved under the conditions characterized by the addition of Pluronic F-68, inoculation at an initial cell density of 3.5 × 105 cells/mL and harvest of infected cells at 48 h post infection (hpi). This material was used for the evaluation of the ATPS recovery processes. It was demonstrated that the chemical components of the ATPS did not have a significant effect upon the infectivity of the adenoviral vectors and a total recovery of approximately 90% was obtained. These findings contribute to the process development for the manufacture of adenoviral vectors and other nanoparticulate bioproducts. |
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