Efficient clinical-grade ?-retroviral vector purification by high-speed centrifugation for CAR T cell manufacturing

?-Retroviral vectors (?-RV) are powerful tools for gene therapy applications. Current clinical vectors are produced from stable producer cell lines which require minimal further downstream processing, while purification schemes for ?-RV produced by transient transfection have not been thoroughly investigated. We aimed to develop a method to purify transiently produced ?-RV for early clinical studies. Here, we report a simple one-step purification method by high-speed centrifugation for ?-RV produced by transient transfection for clinical application. High-speed centrifugation enabled the concentration of viral titers in the range of 10(7)-10(8) TU/ mL with >80% overall recovery. Analysis of research-grade concentrated vector revealed sufficient reduction in productand process-related impurities. Furthermore, product characterization of clinical-grade ?-RV by BioReliance demonstrated two-logs lower impurities per transducing unit compared with regulatory authority-approved stable producer cell line vector for clinical application. In terms of CAR T cell manufacturing, clinical-grade ?-RV produced by transient transfection and purified by high-speed centrifugation was similar to ?-RV produced from a clinical-grade stable producer cell line. This method will be of value for studies using ?-RV to bridge vector supply between early- and late-stage clinical trials.

Automated summary

A simple one-step purification method using high-speed centrifugation was developed to purify transiently produced ?-RV for clinical application. The method achieved concentration of viral titers in the range of 10(7)-10(8) TU/mL with >80% overall recovery. Purification of ?-RV using this method resulted in significantly lower impurities compared to stable producer cell line vectors approved for clinical application.