Development And Validation Of A Quantitative Method For Determination Of CD19-targeted Chimeric Antigen Receptor T Cell In Pharmaceutical Products

Chimeric Antigen Receptor T(CAR-T)cell is a type of genetically engineered T cell that express an artificial receptor to target a specific protein and activate T cell function.Numerous clinical trails demonstrated that adoptive transfer of CAR-T cells is a promising therapy for B-cell-derived cancers.Taking CAR-T cells from the labs to the patients requires sophisticated pharmaceutical production and quality control capability.In this study,on the basis of the development of a novel anti-FMC63antibody(R19),a flow cytometry method for the quantitative determination of Anti-CD19-CART(FMC63 scfv)cells and its CD4/CD8 subtypes were developed and optimized.The method validation was carried out in accordance with relevant ICH guidelines.This study was also extended to investigate the relationship between variables in experimental condition and data output of the flow cytometry method.The study demonstrated that the method with the novel antibody,R19 was not only highly specific for Anti-CD19-CART cell determination but was superior to those with conventional reagents in the separation between CAR-positive population and CAR-negative population.We also identified that cell debris and dead cells contributed to false positive or negative signal in the flow cytometry detection.Excellent linearity(R~2>0.99)was also obtained over concentration ranges of(10-90%),(10-90%)and(5-80%)for CD3+CAR+,CD4+CAR+and CD8+CAR+,respectively.The parameters of accuracy and precision indicated this method with parapllel prepared calibration curve have fulfilled the requirements in Bioanalytical Method Validation Guidance for Industry(by FDA).Applying Design of experiment(DOE)approach allowed us to systematically investigate impact of variables in experiment condition on metrics of the method.5 out of 6 models established by fractional factor analysis pointed out that the factor of cell count in sample exerted the most profound influence on data output.This result was verified by a challenge test.In summary,we developed and validated a novel FC method that would play a pivotal role in quality control system of a pharmaceutical degree Anti-CD19-CART cell product.