GMP-translatable culture vessels improve manufacturing outcomes
The SNBTS second-generation EBV-specific T cell bank was manufactured in standard culture flasks, and we determined whether transfer to more GMP-compliant G-Rex culture flasks would affect T cell phenotype or functional markers. Sero-positive apheresis material from healthy EBV-positive donors (n=6) were used to establish fresh EBV-specific T cell cultures, and each donor culture was split to directly compare culture in closable G-Rex flasks (G-Rex100) versus standard tissue culture flasks (Corning) through an optimized three-round stimulation process. The G-Rex flasks used had a standard research cap, but are identical to GMP-compliant flasks which have a closed-process cap, so can be considered GMP-translatable. At day 30 (9 days after the third LCL stimulation), cells were harvested from both culture vessels and analyzed by surface and intracellular cytokine phenotyping. The mean percentage of CD8+ T cells was significantly higher (p=0.012) in G-Rex flask than in standard flask (90.2 ± 3.49% versus 78.4 ± 2.11% respectively, Figure 2A). However, T cell surface marker phenotype did not differ significantly between culture flasks (Figure 2B). Intracellular cytokine analysis after PMA/ionomycin stimulation demonstrated that there was no significant difference in either quantity of cytokine expression (measured by cMFI, Figure 2C) or cytokine co-expression subpopulations in CD8+ T cells between flasks (Figure 2D). These results demonstrate using manufacturing-scale G-Rex culture flasks not only allows increased cell expansion (data not shown), but also develops a more consistent T cell phenotype in a shorter period of time than culture in conventional Corning flasks. This process is then readily transferable to G-Rex closed-system flasks to allow a fully closed manufacturing process via use of the Gatherex cell harvester (Wilson Wolf Ltd).