Cell therapy is widely carried out around the world, and the main types of cell therapy are stem cell therapy and immune cell therapy. In the direction of immune cell therapy, the research and development of CAR-X (CAR-T, CAR-NK, etc.) products that have undergone genetic modification are the hottest. Taking CAR-T as an example, as of now, there are more than 800 CAR-T cell therapy drugs under research worldwide, of which 8 have been approved for marketing. Domestically, as of March 9, 2022, CDE has accepted a total of 135 applications for cell therapy products, of which 70 are CAR-T drugs. There are 2 CAR-T products approved for marketing. Cell therapy products and technical products modified by genetic modification are more popular.
CAR-T, the full name is chimeric antigen receptor T-cell immunotherapy, that is, chimeric antigen receptor T cell immunotherapy. Compared with general immune cell therapy products, it is characterized by genetic modification and transformation with specific CAR. CAR gene transduction is a key step in the process of CAR-X products, and it is also the core technology. Generally speaking, CAR gene transduction methods mainly include lentiviral and retroviral transduction or electroporation. Among them, electroporation technology is currently the most studied non-viral vector system, but current electroporation methods usually produce poor cell viability and relatively low transfection efficiency. Retroviral vectors are the first viral vectors that are widely used clinically, but because retroviral vectors can only transduce non-quiescent T cells, lentiviral vectors can transduce both non-quiescent and quiescent T cells. Cells, with higher safety, is currently the most widely used gene transduction carrier in CAR-T technology.
Among the 8 CAR-T cell therapy products approved for marketing in the world, 5 CAR-T cells are produced by lentiviral transduction, including Kymriah from Novartis, Breyanzi from Juno/BMS, Celgene/BMS, and WuXi Juno Ricci Orenza Injection and Carvykti of the Legendary Creature. The production process of lentiviral vectors can generally be divided into two parts: upstream and downstream. The upstream process includes cell culture and virus packaging. In upstream production, HEK293T or HEK293FT are usually used as packaging cells. Packaging cells can be cultured in two ways: adherent and suspension:
1. Adherent HEK293T cell culture system: it is the most mature and extensive lentivirus packaging method, with simple process operation and high lentivirus titer. However, it faces serious challenges in terms of safety and large-scale production:
● Using culture medium containing animal-derived serum (FBS) is costly and has uncertain components, which is not conducive to downstream lentivirus purification and is not suitable for clinical application;
● Adherent 293T cells are cultured in culture flasks and cell factories, so it is difficult to scale up the process and increase the output;
● During the process of packaging lentivirus in adherent 293T cells, cells and plasmids are in a static state, and insufficient contact may easily lead to large batch-to-batch differences in virus packaging.
2. Suspension HEK293T cell culture system: Based on the above factors, this field is prompted to develop a suspension technology that is conducive to large-scale production. Compared with the wall-attached HEK293T system, the suspension culture system is easier to scale up, and the process technology is simple and does not require Cultivation in containers treated with tissue culture can simplify the downstream process and reduce the risk of contamination, reduce labor force and production space, and greatly reduce the cost of lentivirus preparation. The composition of the medium is clear, which is more suitable for clinical application. However, at present, the suspension culture system is used for the production of lentivirus packaging. The technology is immature, and most of them are in the process of research and development.
When the initial seeding density was 0.3×10^6 cells/mL, it expanded to over 3.08×10^6 cells/mL within 72 hours, and the cell expansion was more than 10 times. The average doubling time was less than 24 hours, and the stability was good.
3.3. Comparative test of lentivirus production yield