Practical Experience with Automated Technologies in Anti-CD19 CAR T-Cell Product Manufacturing

Chimeric antigen receptor (CAR) T-cell therapy has transformed the treatment of relapsed or refractory hematological malignancies. However, conventional CAR T-cell manufacturing is labor-intensive and costly, faces challenges in scaling production and carries a potential risk of contamination, supporting the adoption of closed, automated systems for point-of-care manufacturing. Here, we performed a functional comparative characterization of CD19 CAR T-cell products generated through an extended manufacturing cycle on the Wukong (SinoBiocan, China) and KunLun (XinBio, China) automated platforms using a single leukapheresis product. Both platforms generated viable T-cell products with high post-selection purity and measurable CAR expression. On day 10, the KunLun-produced CAR T product yielded 5.11 × 108 cells (99.4% CD3+), with 23.9% CAR expression (approximately 1.22 × 108 CAR+ cells), a balanced CD4:CD8 ratio (approximately 1:1) and >90% cell viability. The Wukong product yielded 2.64 × 108 cells (98.3% CD3+), with 18.5% CAR expression (approximately 5.0 × 107 CAR+ cells) and a CD4-skewed subset distribution, also with >90% viability. In a fluorescence-based coculture assay using the human Burkitt’s lymphoma-derived cell line Raji-mKate2 as a target, both products demonstrated CD19-specific cytotoxicity. KunLun-derived CAR T cells exhibited higher cytolysis rates across the tested effector-to-target ratios, reaching 78% specific lysis at 10:1 after 24 hours. Together, these data support the feasibility of closed, automated point-of-care CAR T-cell manufacturing and indicate that platform design and process implementation can influence final yield, CAR+ cell output, product composition and functional activity. Since the current analysis was performed using leukapheresis material from a single donor, future studies using additional donors, replicate runs and deeper product profiling are needed to identify parameters that best predict the clinical performance of CAR T-cell products. PEER REVIEWED ARTICLE **Peer reviewers:** Prof. Ancha Baranova, School of Systems Biology, George Mason University, Fairfax, Virginia, USA One anonymous peer reviewer Received on November 21, 2025; accepted after peer review on March 30, 2026; published online on April 09, 2026.