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Zhang, Yishuang

University of British Columbia

Chimeric antigen receptor (CAR) T-cell therapy has emerged as a highly promising cell-based approach for the treatment of cancers. A key initial step in biomanufacturing is for T-cells to be activated before their transduction so as to promote their transfection and proliferation into high yields of CAR T-cells. Soluble activators provide an attractive alternative for biomanufacturing since they eliminate the cell losses when magnetic beads are removed, and this also simplifies bioprocessing. However, understanding the soluble activator exposures of human T-cells is a potential critical process parameter in the biomanufacturing of CAR T-cells. As alternative functional assays were assessed, it was found that upon activation, a major change in cell size was observed long before the cell numbers increased. Thus, a functional assay to measure the activity in culture supernates was developed based on measurements of T-cell diameters. Image analysis was used to measure the sizes of T-cells after 24, 48, or 72 hours of culture to determine the duration that most effectively distinguished variable activity levels. This activity assay was then used to determine the soluble activator levels over time, as well as to distinguish mechanisms of activity loss caused by binding to culture vessel surfaces, thermal degradation, or cell binding/internalization. The cell binding/internalization was by far the greatest contributor to activity decreases. Thus, this assay provides a means to monitor a key variable so as to increase the process understanding of CAR T-cell biomanufacturing. Additionally, the feasibility of refreezing many small T-cell aliquots with an acceptable recovery rate was demonstrated.

Text

2023-10-19

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/86214

Biomedical Engineering

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/