The Future of Distributed Manufacturing: Rapid Production and Release of CAR-T on a Microfluidic Chip

Abstract

Background & Aim

Chimeric Antigen Receptor-T (CAR-T) cell therapy is a powerful new tool in the fight against cancer, with approved products for treatment of cancer and promising results in non-malignant diseases. While CAR-T has shown clinical utility, its complex manufacturing process results in high costs, with the price of a single dose greater than $350k. The manufacturing process relies on centralized production in specialized cGMP clean rooms using highly trained personnel. Significant overhead and logistics are required to handle multiple patient samples at once and ensure transportation of patient material between treatment and manufacturing sites.

We have developed a microfluidic chip-based approach that can integrate all critical process elements of CAR-T manufacturing (cell isolation, washing, culture, characterization and release testing) into a closed system. Critical design features allow cost-effective optimization and validation of new protocols from research scale to clinical production. We demonstrate the utility of this approach by making an antiCD19 CAR-T product.

Methodology

A microfluidic device was created with 310k microwells with 30 μm diameter and 100 μm depth. AntiCD19 CAR-T cells were produced by capturing CD3 cells into microwells from PBMCs using positive immunomagnetic isolation. Cells were labeled with CD3 and CD45 surface markers to characterize purity and total cell loading. Following characterization, perfusion culture was performed with T cell culture media and an anti-CD19 lentiviral vector (LVV) that also transduces GFP for 18 hours. After culture, the chip was imaged to characterize percentage of transduced cells by GFP expression. Export was performed by inverting the chip to sediment cells from wells and flushing to a collection vial. The entire process takes less than 24 hours.

Results

In three experiments, an average of 3.7e6 cells were loaded with 99.8% CD3 purity. After 18 hours of culture with LVV, 57% of cells were CAR+ as measured by GFP expression. Images of the chip before and after export indicate 2.61e6 cells were exported with post collection viability averaging 90%.

Conclusion

We have demonstrated an integrated, closed CAR-T manufacturing process capable of producing millions of cells in less than 24 hours. Our approach is scalable to produce larger doses has the potential to significantly reduce costs in a format that is easy to scale from concept to clinic.