A 3-in-1 integrated automated platform for rapid CAR-T cell manufacturing: activation, transduction, and expansion in a hollow-fiber system.

Abstract

Modifying a patient's own immune system to treat oncologic and autoimmune diseases using CAR-T therapy has become a major focus of research and clinical studies. While these therapies have immense capacity to treat and even cure diseases on a large scale, barriers such as complex manufacturing processes and long turnaround time impede widespread access. Traditional CAR-T manufacturing involves manual and open steps of activation and viral transduction before expansion, causing inefficiencies, inconsistencies, and extended timelines. A fast and simplified GMP-compliant manufacturing process for high quality CAR-T cells is needed to support broader access and adoption of these therapies. This article demonstrates the successful integration of 3 steps (activation, virus transduction, and expansion) into 1 automated and functionally closed Quantum™ Cell Expansion System platform, thereby named 3-in-1. PBMCs from 3 healthy donors were isolated, followed by CD3+ T cell enrichment. A total of 1.2 × 10⁸ CD3+ T cells were seeded into the Quantum system for automated activation, viral transduction, and expansion in serum-free culture conditions. The harvested cells were washed and concentrated using CellSep PRO, with quality and functionality evaluated via flow cytometry and a non-radioactive cytotoxicity assay. The Quantum system produced over 1.2 × 10¹⁰ cells (117 ± 14 fold expansion) within 7 to 8 days, with on average 44.9 ± 4.0% viral transduction. The manual flask control was able to reach 100-fold expansion from Day 10 with an average transduction efficiency of 25.6+. The cells harvested from Quantum also demonstrated strong cytotoxic activity against NALM-6 cells and a high proportion of T_SCM cells. By providing a concentrated and stable microenvironment, Quantum 3-in-1 simplifies CAR-T workflow, shortens manufacturing cycles, enhances viral transduction efficiency, and generates clinically optimal cell phenotypes.