Closed, Automated CAR-T Cell Manufacturing: from Research to Point of Care production.

Abstract

Background: Point-of-care manufacturing of CAR-T cells could reduce cost and improve patient access, but few closed, automated systems support both flexible process development and GMP production. Methods and results: We describe LimCORE, a single-use consumable for cell incubation and processing, implemented in LimGROW (manual system) and LimONE (closed and automated system). In LimGROW, T cells expanded 246 +/-18 fold over 10 days at optimized seeding density, versus 89 +/-9 fold in a G-Rex control, with similar viability (>90%) and phenotype. Process parameters directly transferred to LimONE, where automated, closed buoyancy-based CD3+ selection achieved 90.3 +/-3.5% purity and 59.2 +/-17.0% recovery in under two hours. Using this workflow, we manufactured CD19 CAR-T cells in a fully closed, automated 7-day LimONE process requiring 155 minutes of operator intervention. LimONE manufactured CAR-T cells reached 42.6 +/-2.4 fold expansion (532 +/-29x10e6; cells at harvest), with 43.6 +/-6.0% transduction efficiency and viability similar to G-Rex controls. CD4+/CD8+ ratios, CD8+ differentiation subsets, and cytotoxicity were comparable between platforms, while LimONE-manufactured cells showed increased spare respiratory capacity and oxygen consumption. Across four manufacturing runs on the LimONE, no hardware or software failures occurred, and automated liquid transfers and volume concentration stayed within +/-5% accuracy. Conclusions: These data show that LimCORE supports T cell expansion, selection, and complete CD19 CAR-T manufacturing with performance similar to existing platforms while reducing operator time, across R&D and GMP scale closed-system formats.