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 vectorproducing 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 À801C 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 nonhuman 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. Gene Therapy (2006) 13, 95-100.

Multifactorial Optimization of Gammaretroviral Gene Transfer into Human T Lymphocytes for Clinical Application

“…cGMP-grade PG13-1928z1 vector stocks were prepared as previously described (Przybylowski et al, 2006). Briefly, PG13-1928z1 cells were initially seeded from one certified master cell bank (MCB) cryovial, containing 10 7 cells, and ultimately expanded into four 10-tray Nunclon Cell Factories (Nunc brand; Thermo Fisher Scientific, Rochester, NY).…”

Generation of a high-titer packaging cell line for the production of retroviral vectors in suspension and serum-free media

“…15,21,25,[27][28][29][30][31] On the contrary, a packaging cell line that could grow in suspension would allow vector production in bioreactors with practically no size limit, and with higher cell densities compared to adherent cells that are limited to growth surface. 29,32 Several years ago, the production of retroviral vectors from human lymphoid cells that naturally grow in suspension was reported.…”

Quantitative analysis of clinically relevant mutations occurring in lymphoid cells harboring γ-retrovirus-encoded hsvtk suicide genes

“…High-titer clones were identified by Southern blot analysis and by fluorescence-activated cell sorting analysis. The PG13-NITcl.2 clone was selected for clinical applications based on its ability to efficiently transduce primary peripheral blood lymphocytes 28 and harbors one copy of the vector as determined by Southern blot (Supplementary Figure S5). The PG13-NG1 bulk population was enriched up to 99% NTP + using LS columns (Miltenyi Biotec GmbH, Auburn, CA, USA) with the 20.4…”