Fig. 1.

Schema of the Sepax spinoculation manufacturing process. Once the apheresis product is collected it was loaded on the Clinimacs Plus to be selected for CD4 + /CD8 + T-cells (simultaneous double positive selection). On day 0, the selected cells were incubated with anti-CD3/CD28 Dynabeads at a ratio of 3:1 (beads:cell) with a 40 IU of IL-2. These cells are incubated for 48 h before transduction. On day 2, the cells are divided into three different groups for the transduction process. For the static transduction, cells and vector were added to culture bags (Origen). Sepax spinoculation involves the cells, media, and vector be loaded to separate transfer packs and culture bags. The instrument and kit loads and washes the cells before the vector is added. The cells and vector are spun at a speed of 1000×g for 1 h. After spinoculation, cells are loaded into a culture bag (Origen). The bag spinoculation method is similar to the Sepax method, but the cells and vector are loaded into a culture bag (Origen), placed in an overwrap bag, and then loaded into the centrifuge buckets where they are spun at 1000×g for 2 h. In all methods the cells are expanded until day 9. On day 4, the Dynabeads are removed and discarded using a DynaMag CTS. On day 9 the cells are washed, assayed and cryopreserved. Cells may be thawed for functional testing, or if a clinical product, prepared for infusion in patients