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      Large-scale production of lentiviral vectors using multilayer cell factories

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          Abstract

          Lentiviral-mediated gene therapy has been proposed for the treatment of a range of diseases, and due to its genome integration properties, it offers the potential for long-lasting benefit from a once-off treatment. Production methods for pre-clinical studies in animal models, and ultimately for human clinical trials, must be capable of producing large quantities of high-quality lentiviral vector in an efficient and cost-effective manner. We report here a medium-scale method (from 1.5 L to 6 L of vector supernatant) for lentiviral vector production in adherent cell cultures using the NUNC™ EasyFill™ Cell Factory™ from Thermo Fisher Scientific. Downstream purification uses a Mustang Q XT5 anion exchange capsule from Pall, and an ultracentrifugation step to concentrate the vector. This method is capable of producing lentiviral vector with concentrated titres of 10 8–10 9 TU/ml, with reduced manual handling compared to single monolayer flask methods.

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          Most cited references14

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          Production of lentiviral vectors

          Lentiviral vectors (LV) have seen considerably increase in use as gene therapy vectors for the treatment of acquired and inherited diseases. This review presents the state of the art of the production of these vectors with particular emphasis on their large-scale production for clinical purposes. In contrast to oncoretroviral vectors, which are produced using stable producer cell lines, clinical-grade LV are in most of the cases produced by transient transfection of 293 or 293T cells grown in cell factories. However, more recent developments, also, tend to use hollow fiber reactor, suspension culture processes, and the implementation of stable producer cell lines. As is customary for the biotech industry, rather sophisticated downstream processing protocols have been established to remove any undesirable process-derived contaminant, such as plasmid or host cell DNA or host cell proteins. This review compares published large-scale production and purification processes of LV and presents their process performances. Furthermore, developments in the domain of stable cell lines and their way to the use of production vehicles of clinical material will be presented.
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            Development of a scalable process for high-yield lentiviral vector production by transient transfection of HEK293 suspension cultures.

            Lentiviral vectors (LV) offer several advantages over other gene delivery vectors. Their potential for the integration and long-term expression of therapeutic genes renders them an interesting tool for gene and cell therapy interventions. However, large-scale LV production remains an important challenge for the translation of LV-based therapeutic strategies to the clinic. The development of robust processes for mass production of LV is needed. A suspension-grown HEK293 cell line was exploited for the production of green fluorescent protein-expressing LV by transient polyethylenimine (PEI)-based transfection with LV-encoding plasmid constructs. Using third-generation packaging plasmids (Gag/Pol, Rev), a vesicular stomatitis virus G envelope and a self-inactivating transfer vector, we employed strategies to increase volumetric and specific productivity. Functional LV titers were determined using a flow cytometry-based gene transfer assay. A combination of the most promising conditions (increase in cell density, medium selection, reduction of PEI-DNA complexes per cell, addition of sodium butyrate) resulted in significantly increased LV titers of more than 150-fold compared to non-optimized small-scale conditions, reaching infectious titers of approximately 10(8) transducing units/ml. These conditions are readily scalable and were validated in 3-liter scale perfusion cultures. Our process produces LV in suspension cultures and is consequently easily scalable, industrially viable and generated more than 10(11) total functional LV particles in a single bioreactor run. This process will allow the production of LV by transient transfection in sufficiently large quantities for phase I clinical trials at the 10-20-liter bioreactor scale.
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              Challenges of up-scaling lentivirus production and processing.

              Lentiviruses are becoming an increasingly popular choice of gene transfer vehicle for use in the treatment of a variety of genetic and acquired human diseases. As research progresses from basic studies into pre-clinical and clinical phases, there is a growing demand for large volumes of high purity, concentrated vector, and accordingly, the means to produce such quantities. Unlike other viral vectors, lentiviruses are difficult to produce using stable cell lines, therefore transient transfection of adherent cell lines is conventionally used, and this method has proven challenging to up-scale. Furthermore, with the required increases in the volume of vector needed for larger animal and human use, comes the need for more efficient and sophisticated supernatant purification and concentration techniques. This review presents the challenges of up-scaling lentivirus production and processing approaches, novel systems for overcoming these issues, and the quality assessments recommended for producing a clinical grade lentiviral gene therapy product.
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                Author and article information

                Journal
                J Biol Methods
                J Biol Methods
                Journal of Biological Methods
                Journal of Biological Methods
                2326-9901
                2018
                10 April 2018
                : 5
                : 2
                : e90
                Affiliations
                Department of Respiratory and Sleep Medicine, Women’s and Children’s Hospital , 72 King William Road, North Adelaide SA 5006, Australia
                Robinson Research Institute , Adelaide SA 5000, Australia
                Adelaide Medical School, University of Adelaide , Adelaide SA 5005, Australia
                Author notes
                *Corresponding author: Nathan Rout-Pitt, Email: nathan.rout-pitt@ 123456adelaide.edu.au

                These authors contributed equally to this work.

                Competing interests: The authors have declared that no competing interests exist.

                Abbreviations used: BSA, bovine serum albumin; CFTR, cystic fibrosis transmembrane conductance regulator, FCS, fetal calf serum; HEK, human embryonic kidney; LV, lentiviral; PBS, phosphate buffered saline; PEI, polyethylenimine; RT- PCR, reverse transcription- polymerase chain reaction

                Article
                10.14440/jbm.2018.236
                6706103
                31453241
                325c6935-85ee-40b9-8657-45dda6aa30fa
                © 2018 The Journal of Biological Methods, All rights reserved.

                This work is licensed under a Creative Commons Attribution 3.0 License.

                History
                : 02 November 2017
                : 01 March 2018
                : 14 March 2018
                Page count
                Figures: 3, Tables: 2, Equations: 0, References: 20, Pages: 9
                Categories
                Protocol

                calcium phosphate,cell factories,lentiviral vector,mustang q,ultracentrifugation

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