Journal Article

Production scale-up and validation of packaging cell clearance of clinical-grade retroviral vector stocks produced in Cell Factories

Hakakha, A, Przybylowski, M, Stefanski, J, Sadelain, M, Hodges, J, Rivière, I

2006

Overview

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.

• Publisher: Nature Publishing Group UK,Nature Publishing Group
• Subject: Anesthesia. Intensive care medicine. Transfusions. Cell therapy and gene therapy / Animals / Applied cell therapy and gene therapy / Biological and medical sciences / Biomedical and Life Sciences / Biomedicine / Bioreactors / Biotechnology / Cell Biology / Cell lines / Cell Separation - methods / Freezing / Fundamental and applied biological sciences. Psychology / Gene Expression / Gene Therapy / Genetic Therapy - methods / Genetic Vectors / Health. Pharmaceutical industry / Human Genetics / Humans / Industrial applications and implications. Economical aspects / Industry / Medical sciences / Methods / Nanotechnology / Physiological aspects / Plasmids / Production processes / Quality Control / Retroviridae - genetics / short-communication / Transfusions. Complications. Transfusion reactions. Cell and gene therapy