Table 2:

Quality Control assessment of three batches of humanized CD19 CART-cells manufactured in cGMP compliant settings

Quality Control of Humanized CD19 CART-cells
Criteria	Parameter	Method	Acceptance criteria	Batch 1	Batch 2	Batch 3
Safety	RCL test	VSV-G gene qPCR: Taqman based detection	Negative for VSV-G gene	Negative	Negative	Negative
	Sterility	BACTEC and culture based assay	Negative	Negative	Negative	Negative
	Mycoplasma	PCR based	Negative	Negative	Negative	Negative
Identity	Appearance	Visual inspection	Yellowish, Milky, no aggregates in cell suspension	Milky, no aggregates	Yellowish, no aggregates	Yellowish, no aggregates
	CAR surface expression	% protein L positive of viable CD3+ T cells	Transduction efficiency (TE)- ≥ 10%	23%	33%	30%
	CAR copy number (copies/ 100ng DNA)	Taqman based CAR gene qPCR method	CAR gene detection with > 5 copies	100 copies	100 copies	100 copies
Purity	Cell viability	Trypan blue exclusion assay	Cell viability > 70%	91%	96.3%	94%
	CD19 + viable cell detection	CD19 protein surface detection by Flow cytometry	Negative for the presence of CD19+ cells	Negative	Negative	Negative
	Detection of magnetic beads	Microscopic detection of CD3/CD28 magnetic beads	Presence of residual beads (<100 beads/3×10^6 cells)	No beads	57 beads/3×10^6 cells	16 beads/3×10^6 cells
Potency	Cytokine detection	IFN-γ ELISA assay	IFN-γ release (>50pg/ml)	Detected	Detected	Detected
	Cytotoxicity	Flow cytometry based	>50% killing of CD19+ tumor cells	Achieved	Achieved	Achieved