Objectives: Evaluating the quality of CAR-T cells for CAR-T cell therapy in vitro treats acute lymphoblastic leukemia.
Research subjects and methods: CAR-T CD19RCD137/pMC and CAR-T CD19RCD137- iCasp9-IL15/pMC cells were cultured to determine sterility. The cell suspension sample product was tested the endotoxin limit standard. Then, the protein was extracted by SDS page electrophoresis and western blotting. The albumin standard curve was constructed and E. coli protein was quantified using a Western Blot analysis system. Evaluating the expression of E. coli mRNA using real-time PCR.
Results: Endotoxin detection limit: 0.005-0.5 EU/ml, endotoxin concentration < 0.045 EU/ml. CAR-T cells were sterile. Establishing the standard curve with 1 ml of culture fluid containing 352.585 µg of protein. The weight of total protein residue in 1 ml of culture fluid is 1.15 mg, the total protein content accounts for 3.3%. The proportion of E. coli protein in the culture fluid sample of CAR-T cells is 0.06-0.21%. The culture fluid of CAR T cell samples at 3, 7 and 14 days did not show E. coli.
Conclusion: The results of bacterial culture, quantification of endotoxin and E. coli protein showed that the CAR-T cell product met the quality standard.