Development and Validation of a Quantitative LCMS/MS Method for Measuring CYP4V2 Enzyme Activity in rAAV-hCYP4V2 Gene Therapy Products

Bietti crystalline dystrophy (BCD) is a hereditary retinal disease caused by loss-of-function mutations in the CYP4V2 gene. Gene replacement therapy using rAAV-hCYP4V2 represents a promising therapeutic strategy, requiring robust bioassays for product quality control. This study developed and validated a sensitive LC-MS/MS method for quantifying CYP4V2 enzyme activity. Lysates from HeLa-AAVR cells transduced with rAAV-hCYP4V2 (MOI=3×105) were used, with lauric acid as substrate supplemented with cytochrome P450 reductase, cytochrome b5, and NADPH. The ω-hydroxylated product (12-hydroxy lauric acid) was quantified using tolbutamide as internal standard. Method validation followed ICH guidelines. Results demonstrated excellent specificity with negligible background in negative controls. Linearity was achieved over 0.5-100 ng/mL (R2>0.99), with average recovery of 100.6%. Intra-batch and inter-batch precision RSDs were <47.8% and <28.4%, respectively. Product stability was maintained ≥4 weeks at -80℃. The method was successfully applied to three AAV serotypes (AAV2, AAV8, AAV2/8), with all RSDs <23.9%. This validated LC-MS/MS bioassay provides a crucial quality control tool for potency assessment, process development, batch release, and stability studies of rAAV-hCYP4V2 gene therapy products.