Abstract
The clinical implementation of gene therapy requires large-scale production of viral vector stocks (VS) derived from packaging cell lines. Upon scaling-up, maintenance of high viral titers and filtration of the VS become significantly challenging. Thus, production schemes amenable to straightforward validation must be developed. To this end, we have established a semi-closed process to manufacture batches of 7 l or more of clinical-grade oncoretroviral VS using 10-tray Cell Factories. Using a peristaltic pump, the VS are collected on 3 consecutive days, filtered, pooled and stored frozen. To ensure the absence of viable vector-producing cells (VPCs) from each VS unit-dose, we undertook an orthogonal log-removal validation study to demonstrate the ability of both the filtration system to remove viable cells and the VS freezing process to inactivate them. We demonstrate a total VPC-reduction of 11.6 log, thus insuring the absence of contaminating VPCs in transduced clinical samples. We also show that this production process generates stable VS that can be stored at −80°C for more than 3 years. Importantly, this relatively simple and affordable process can be customized to generating large volume of VS for small animal or non-human primate studies. This methodology is not limited to the generation of cell-free clinical oncoretroviral VS, and can be applied to other types of vectors produced in packaging cell lines, such as lentiviral vectors.
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Acknowledgements
This work is supported by US National Institutes of Health Grants CA-59350, CA-08748, HL66952 and by Mr William H Goodwin and Mrs Alice Goodwin and the Commonwealth Cancer Foundation for Research & the Experimental Therapeutics Center of MSKCC.
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Przybylowski, M., Hakakha, A., Stefanski, J. et al. Production scale-up and validation of packaging cell clearance of clinical-grade retroviral vector stocks produced in Cell Factories. Gene Ther 13, 95–100 (2006). https://doi.org/10.1038/sj.gt.3302648
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DOI: https://doi.org/10.1038/sj.gt.3302648
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