January 27-31, 2019
The field of immunotherapy has emerged as a promising new type of treatment for cancer with the approval of the first two CAR-T therapies. The clinical success of T-cell based immunotherapies necessitates a robust manufacturing process for these products to be consistently produced at commercial scale. Our CAR-T workflow combines unit operation specific solutions for thaw of an apheresis unit, wash, CD3 selection, T-cell activation, lentiviral transduction, incubator- and reactor-based expansion culture, harvest, formulation, cryopreservation and thaw of CAR-T product. We have evaluated the impact of both serum-containing and xeno-free culture media, commercially available T-cell selection and activation reagents, closed small-scale culture vessel options, alternative solutions to enhance transduction, and the specific timing of process steps to develop a modular platform process that is robust and flexible for the varied needs of CAR-T developers. Frozen apheresis units are processed using the SmartWash protocol on the SepaxTM 2 and T-cells are isolated with EasySepTM Release CD3 Positive Selection Kit. The cells are then activated with ImmunoCult CD3/CD28/CD2 T-cell activator before being transduced 24 hours later using the SepaxTM 2. Expansion of Tcells are carried out in two stages: incubator-based culture before going into the XuriTM Cell Expansion System W25 with a perfusion feeding regime. Cultured cells are then harvested and washed in Plasmalyte-A with human serum albumin and formulated with CryoStor® CS10 using the FlexCell protocol on the Sefia™ Cell Processing System. The final cell products are cryopreserved using the VIA Freeze controlled-rate freezer. We have also accessed a point-of-care thawing strategy using the VIA Thaw. Our CAR-T process achieves greater than 1.0E10 expanded T-cells with >80% eGFP transduction efficiency across an 8-day manufacturing process.
Loo-Yong Steven Kee, Calley Hirsch, Devina Ramsaroop, Elizabeth Csaszar, Danylo Sirskyj, and Aaron Dulgar-Tulloch, "Development of a closed CAR-T manufacturing process" in "Advancing Manufacture of Cell and Gene Therapies VI", Dolores Baksh, GE Healthcare, USA Rod Rietze, Novartis, USA Ivan Wall, Aston University, United Kingdom Eds, ECI Symposium Series, (2019). https://dc.engconfintl.org/cell_gene_therapies_vi/33