MANUFACTURING PROCESS IMPROVEMENT FOR SCALE UP OF AN AUTOLOGOUS ADOPTIVE T CELL PRODUCT

• Published in: Cytotherapy (Oxford, England) England), 2024-06, Vol.26 (6), p.S138-S138
• Main Authors: Missan, D.S., Reckard, G.A., Pathangey, L.B., Byrd, T., Herring, J., Bergsagel, L., Gustafson, M.
• Format: Article
• Language: English
• Subjects: process development; process improvement; Scale Up
• ISSN: 1465-3249; 1477-2566
• DOI: 10.1016/j.jcyt.2024.03.266

Improving a GMP manufacturing process is an important goal of a cell therapy lab. We have established an autologous adoptive T cell platform which involves the expansion of antigen specific T cells. While successful in manufacturing products for the first 5 patients in our phase 1 clinical trial, we have identified several key manufacturing steps to optimize. Additionally, future clinical trials that will utilize this manufacturing platform require even larger cell numbers. Here we demonstrate development of processes to both scale up our cell manufacturing and utilize both automation and modification of steps in the manufacturing process. We aimed to assess culturing these T cells in larger vessels, as well as potential freezing strategies for our final products while determining the impact these changes have on our product consistency. PBMCs were isolated from a leukapheresis product from either healthy donors or multiple myeloma patients and cryopreserved. The PBMCs were thawed and seeded into 6-well G-Rex plates, 1L and/or 5L G-Rex vessels followed by stimulation and expansion of T cells. Growth kinetics were monitored in the 6-well plates with cells harvested on days 5, 9, 12, 15 and 19 and evaluated by flow cytometry for CD45, CD3, CD4, and CD8 and viability using 7-AAD. Supernatants were harvested from 6-well plates on days 5, 7, 9, 12, 15, and 19, and from the 1L and 5L cultures on days 7, 9, 12, 15, and 19 and evaluated for metabolites. Final T cells were frozen in vials in either CS5 or CS10. For single cell analysis, cells were thawed and recovered overnight in media. CD8+ and CD4+ cells were magnetically selected. Unstimulated or stimulated cells with either PMA/Ionomycin or CD3/28 were stained and then loaded onto the Adaptive Immune Cell chips, run on the Isolight, and analyzed with the IsoSpeak software. The viability of the final T cell product was minimally affected by the different freezing media, however it was the PSI of the CD4+ T cells upon stimulation was reduced with CS5 media. Additionally, using the same cell seeding density, the 5L vessel resulted in reduced cell numbers. Based on our data, we hypothesize that the larger the vessel, the more time cells need to reach maximum cell capacity. We also postulate that monitoring of lactate during culture will allow the best timing to harvest the final product. Additionally, the different concentrations of DMSO in frozen samples can affect the polyfunctionality of the product.