Integrated T-cell enrichment, static transduction, and expansion in gas-permeable bioreactors for adoptive cell...

Broader access to autologous chimeric antigen receptor (CAR) T-cell therapy is limited by costly, device-bound manufacturing workflows and reliance on dedicated instrumentation for discrete unit operations, including T-cell enrichment and culture. These constraints affect academic, decentralized and high-throughput centralized manufacturing programs. We developed a closed-system CAR T-cell manufacturing workflow centered on a gas-permeable G-Rex bioreactor platform that enables direct in-vessel CD4⁺/CD8⁺ T-cell enrichment, static viral transduction and expansion without requiring a separate standalone enrichment device or spinoculation hardware.

Key unit operations of a G-Rex-based GD2 CAR T-cell manufacturing process were optimized, including RetroNectin-mediated static retroviral transduction, coating concentration and timing, and media-exchange strategy. T-cell phenotype and functional potency were characterized longitudinally throughout the 7-day culture period. The optimized workflow was translated to a manufacturing-scale G-Rex 100M-CS process incorporating a newly developed, previously unpublished method for direct in-vessel CD4⁺/CD8⁺ magnetic enrichment using detachable beads and a prototype SepaRex magnetic base. At-scale products from three healthy donors were assessed for enrichment performance, transduction efficiency, expansion, viability, phenotype, cytokine secretion and tumor-cell killing, and benchmarked against historical products manufactured using a clinically validated CliniMACS Prodigy-based GD2 CAR T-cell process.

Retronectin-coated G-Rex cultures supported efficient static retroviral transduction without spinoculation, and media exchange between 24 and 96 h post-transduction did not significantly affect final yield or viability. Direct in-vessel CD4⁺/CD8⁺ enrichment achieved 61.6 ± 8.6% recovery and 87.2 ± 7.9% purity. At manufacturing scale, the integrated workflow generated GD2 CAR T cells with 62.3 ± 5.1% transduction efficiency, 26.8 ± 4.7-fold expansion, >94% viability and >96% CD3⁺ T-cell purity. Final products retained central memory features, secreted cytokines and mediated cytotoxicity against GD2-expressing tumor targets, with manufacturing performance comparable to the historical Prodigy-based process.

This closed, scalable and cost-conscious G-Rex-based workflow integrates T-cell enrichment, activation, static viral transduction and expansion within a single bioreactor-centered process. By eliminating dedicated enrichment instrumentation and reducing reliance on highly automated, device-bound culture systems, this bioreactor-centered platform may support flexible CAR T-cell manufacturing across academic, decentralized, and high-throughput centralized settings.