The need for γ-retroviral (gRV) vectors with a self-inactivating (SIN) design for clinical application has prompted a shift in methodology of vector manufacturing from the traditional use of stable producer lines to transient transfection-based techniques. Herein, we set out to define and optimize a scalable manufacturing process for the production of gRV vectors using transfection in a closed-system bioreactor in compliance with current good manufacturing practices (cGMP). The process was based on transient transfection of 293T cells on Fibra-Cel disks in the Wave Bioreactor. Cells were harvested from tissue culture flasks and transferred to the bioreactor containing Fibra-Cel in the presence of vector plasmid, packaging plasmids and calcium-phosphate in Dulbecco's modified Eagle's medium and 10% fetal bovine serum. Virus supernatant was harvested at 10-14 h intervals. Using optimized procedures, a total of five ecotropic cGMP-grade gRV vectors were produced (9 liters each) with titers up to 3.6 × 10(7) infectious units per milliliter on 3T3 cells. One GMP preparation of vector-like particles was also produced. These results describe an optimized process for the generation of SIN viral vectors by transfection using a disposable platform that allows for the generation of clinical-grade viral vectors without the need for cleaning validation in a cost-effective manner.