title: Production scale-up and validation of packaging cell clearance of clinical-grade retroviral vector stocks produced in cell factories.
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 degrees 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.
authors.affiliation: Gene Transfer and Somatic Cell Engineering Facility, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.
authors.forename: M
authors.initials: M
authors.lastname: Przybylowski
authors.forename: A
authors.initials: A
authors.lastname: Hakakha
authors.forename: J
authors.initials: J
authors.lastname: Stefanski
authors.forename: J
authors.initials: J
authors.lastname: Hodges
authors.forename: M
authors.initials: M
authors.lastname: Sadelain
authors.forename: I
authors.initials: I
authors.lastname: Rivière
mesh_terms: D000818:Animals; D019149:Bioreactors; D001709:Biotechnology; D002469:Cell Separation / Q000379:methods; D005615:Freezing; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D007221:Industry; D010957:Plasmids; D011786:Quality Control; D012190:Retroviridae / Q000235:genetics*