Session: 711. Cell Collection and Manufacturing of HSPCs, CAR-T Cells, and Other Cellular Therapy Products: Poster I
Hematology Disease Topics & Pathways:
Emerging technologies, Technology and Procedures
Efficient cell selection using magnetic bead antibody conjugates is a critical step in cell manufacturing for isolating T cells for gene modification. Considering the complexity of blood samples and donor-to-donor variation, washing-off platelet or using Ficoll preparation to separate PBMCs are often the steps prior to magnetic cell selection. However, those steps are time-consuming and very manual. Here we demonstrate a new workflow that integrates the automated, closed immunomagnetic separation platform, MARS, along with GMP reagents and optimized protocols isolating t cells directly from peripheral whole blood as well as leukopak in a timely manner. Our approach simplifies the process, reduces manipulation, thus enhances efficiency and cuts cost. This cell selection process can easily streamline the production of CAR- T used together with a closed system such as G-Rex bioreactors.
Results: T cells were directly isolated from whole blood starting with an initial T cell purity of 22% using CD4/CD8 nanometer magnetic beads. The results show that the positive selection enriched T cell purity to 90%, with a recovery of 57% (average of n=4 samples). We also isolated T cells directly from fresh leukopak starting with an initial T cell purity of 51%, the positive selection process yielded a recovery of 60% T cells with a final purity of 95% (averages of n=12 samples). Enriched T cells showed robust proliferation, expanding over 112-fold by day 10 in culture. Using the new workflow, we also isolated T cells using CD3/CD28 magnetic beads as co-activation reagent achieving a 52% recovery with a purity of 97% from whole blood. Post-isolation, the T cells displayed significant expression of activation markers CD25 and CD69 over a 72-hour period, indicating activation and the functional integrity of the isolated cells. We have demonstrated the cell selection from half leukopak was completed within 90 minutes. We observed no significant impact on cell viability (P<0.5) in any samples.
Conclusion: Our newly developed automated MARS workflow demonstrates scalability for small (million) and large (billion) scale T cell separation with consistent purity and recovery. The workflow simplifies sample preparation, reducing the number of washing steps and providing a time-saving and cost-effective approach. The gentle, column-free magnetic cell separation process maintains cell viability and functionality, making it a new, flexible and robust choice for cell therapy development and manufacturing.
Disclosures: Sa: Applied Cells, Inc: Current Employment. Yu: Applied Cells, Inc: Current Employment.
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