Considerations in the development and validation of real-time quantitative polymerase chain reaction and its application in regulated bioanalysis to characterize the cellular kinetics of CAR-T products in clinical studies.

Real-time quantitative polymerase chain reaction (qPCR) has become the standard method for monitoring cellular kinetics of CAR-T therapies with measurement of the CAR transgene copy numbers in peripheral blood mononuclear cells isolated from patients receiving the treatment. Unlike other biophysical and immunological methodologies for bioanalytical characterization of conventional small molecule drugs or protein biologics, there is no relevant regulatory guidance to date on the method development and validation for quantitative qPCR assays employed during clinical development of CAR-T products. This paper will provide an overview and considerations in the development and validation of a qPCR assay from sample extraction to assay parameters and its implementation in regulated bioanalysis for CAR-T or other types of cell therapies.