An Improved Method for Determining the Infection Titer of Replication-Competent Adeno-Associated Virus

<b>Background/Objectives</b>: Recombinant adeno-associated virus (rAAV) has become a leading vector in gene therapy. However, manufacturing limitations may result in replication-competent AAV (rcAAV) contamination of clinical rAAV products, posing safety risks. Rigorous testing is therefore essential, and the use of accurately calibrated rcAAV reference standard materials is critical for ensuring assay stability and reliability. A disadvantage of the widely used Tissue Culture Infectious Dose 50 (TCID₅₀) assay is its high variability. This study introduces an optimized TCID₅₀ assay for the precise quantification of infectious rcAAV particles. <b>Methods</b>: We developed a TCID₅₀ assay tailored to <i>rep2</i>-based rcAAV, optimizing key aspects such as viral infection conditions, qPCR reaction systems, and standard curve preparation. We employed an innovative strategy to prepare the standard curve using serial dilutions of rcAAV in cell lysate, ensuring alignment with the test sample matrices. <b>Results</b>: The rcAAV-derived standard curve demonstrated exceptional linearity (R² > 0.99), sensitivity (LOQ ≈ 38 copies), and reproducibility, enabling robust endpoint qPCR analysis. The optimized assay significantly improved the precision of the TCID₅₀ assay, as an inter-assay coefficient of variation (CV) of 11.4% was achieved. <b>Conclusions</b>: This refined TCID₅₀ assay is a reliable method for calibrating infectious titers of rcAAV reference standard materials, thereby enabling the standardization of rcAAV testing.