Novel activity assay development for soluble T-cell activators in bioprocessing
department or school: Applied Science, Faculty of
department or school: Biomedical Engineering, School of
data provider: DSpace
degree campus: UBCV
creator: Zhang, Yishuang
issued: 2023-10-19T16:37:36Z
issued: 2023
related degree: Master of Applied Science - MASc
degree grantor: University of British Columbia
description: 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.
aggregated CHO: https://circle.library.ubc.ca/rest/handle/2429/86214
note: NOVEL ACTIVITY ASSAY DEVELOPMENT FOR SOLUBLE T-CELL ACTIVATORS IN BIOPROCESSING by Yishuang Zhang A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF APPLIED SCIENCE in The Faculty of Graduate and Postdoctoral Studies (Biomedical Engineering) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) October 2023 © Yishuang Zhang, 2023 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the thesis entitled: Novel activity assay development for soluble T-cell activators in bioprocessing submitted by Yishuang Zhang in partial fulfilment of the requirements for the degree of MASTER OF APPLIED SCIENCE in Biomedical Engineering Examining Committee: Dr. James M. Piret, Professor, School of Biomedical Engineering, UBC Supervisor Dr. Calvin D. Roskelley, Professor, Department of Cellular & Physiological Sciences, UBC Supervisory Committee Member Dr. Marko Kryworuchko, Adjunct Professor, UBC Chemical and Biological Engineering, UBC Supervisory Committee Member iii Abstract 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 …
has type: Thesis/Dissertation
date issued: 2023-11
is shown at: 10.14288/1.0437232
language: eng
degree discipline: Biomedical Engineering
provider: Vancouver : University of British Columbia Library
publisher: University of British Columbia
rights: Attribution-NonCommercial-NoDerivatives 4.0 International
rights URI: http://creativecommons.org/licenses/by-nc-nd/4.0/
scholar level: Graduate
contributor: Piret, James
type: Text
identifier URI: http://hdl.handle.net/2429/86214