Significant matrix effect in fludarabine quantification in plasma: implications for CAR-T cell therapy monitoring

Event-free survival (EFS) in patients with B-cell acute lymphoblastic leukemia (B-ALL) significantly improves with adequate exposure (as Area Under the Curve: AUC_0-inf or AUC) to fludarabine (FLU) during lymphodepletion prior to chimeric antigen receptor (CAR) T-cell infusion. A cumulative AUC of FLU exceeding 14 mgxh/L is considered optimal, making therapeutic drug monitoring (TDM) crucial for personalized dosing and prolonged CAR T-cell persistence.

We developed and validated an European Medicines Agency (EMA)-compliant high-performance liquid chromatography-tandem mass spectrometry method (HPLC-MS/MS) for FLU TDM, based on similar and published methods, to support clinicians during the patient conditioning phase. All EMA acceptance criteria were met.

Analysis of real plasma samples revealed unexpectedly high FLU concentrations and, consequently, an excessively high AUC. Analysis of plasma samples from a child receiving FLU therapy highlighted the necessity for accurate drug response verification in real matrices. Preparation of quality controls in FLU-free plasma consistently failed without preliminary dilution. Analysis of internal standard range variation enabled more precise and accurate analyses.

This experience highlights the importance of complementing standard validation tests with analyses in each patient's plasma, collected immediately before FLU infusion, to ensure reliable TDM to support CAR T-cell therapy.