Establishing a scalable perfusion strategy for the manufacture of CAR-T cells in stirred-tank bioreactors using a quality-by-design approach.

in Bioengineering & translational medicine by Tiffany Hood, Pierre Springuel, Fern Slingsby, Viktor Sandner, Winfried Geis, Timo Schmidberger, Nicola Bevan, Quentin Vicard, Julia Hengst, Noushin Dianat, Qasim A Rafiq

TLDR

  • A study optimized perfusion parameters for CAR-T cell expansion in bioreactors, increasing yields and quality, and highlighting the importance of scale-down models in reducing time, costs, and risks.

Abstract

Chimeric antigen receptor T cell (CAR-T) therapies show high remission rates for relapsed and refractory leukemia and lymphoma. However, manufacturing challenges hinder their commercial viability and patient accessibility. This study applied quality-by-design principles to identify perfusion critical process parameters for CAR-T expansion in stirred tank bioreactors to maximize yields. A design of experiments in the Ambr® 250 High Throughput Perfusion small-scale bioreactor revealed that earlier perfusion starts (48 h vs. 96 h post-inoculation) and higher perfusion rates (1.0 VVD vs. 0.25 VVD) significantly increased cytotoxic CAR-T cell yields without compromising critical quality attributes. Optimizing perfusion improved growth kinetics and yields across donor samples, achieving densities >21 × 10cells/mL in 7 days, outperforming traditional fed-batch and static flask cultures. This study underscores the importance of optimizing perfusion parameters to maximize CAR-T yields and quality and highlights the utility of scale-down models in reducing time, costs and risks associated with process development.

Overview

  • The study aimed to identify critical process parameters for CAR-T cell expansion in stirred tank bioreactors using quality-by-design principles.
  • A design of experiments was conducted using the Ambr<sup>250</sup> High Throughput Perfusion small-scale bioreactor to optimize perfusion parameters.
  • The study aimed to maximize CAR-T cell yields while maintaining critical quality attributes and improving growth kinetics.

Comparative Analysis & Findings

  • Earlier perfusion starts (48 h vs. 96 h post-inoculation) and higher perfusion rates (1.0 VVD vs. 0.25 VVD) resulted in significantly increased cytotoxic CAR-T cell yields.
  • Optimizing perfusion improved growth kinetics and yields across donor samples, achieving densities >21 × 10<sup>cells/mL</sup> in 7 days.
  • Traditional fed-batch and static flask cultures were outperformed by optimized perfusion cultures in terms of growth kinetics and yields.

Implications and Future Directions

  • Optimizing perfusion parameters is crucial for maximizing CAR-T cell yields and quality.
  • Scale-down models can significantly reduce time, costs, and risks associated with process development.
  • Future studies can focus on further optimizing and validating perfusion parameters for larger-scale bioreactors and clinical applications.
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