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

## TL;DR

This paper presents an improved TCID50 assay for more accurately measuring replication-competent AAV virus, which is important for gene therapy safety.

## Contribution

An optimized TCID50 assay with enhanced precision and reproducibility for rcAAV titer determination.

## Key findings

- The optimized assay achieved a standard curve with R2 > 0.99 and sensitivity of ~38 copies.
- The inter-assay coefficient of variation was reduced to 11.4%, significantly improving precision.
- The method enables reliable calibration of rcAAV reference standards for standardized testing.

## Abstract

Background/Objectives: 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. Methods: We developed a TCID50 assay tailored to rep2-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. Results: 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. Conclusions: This refined TCID50 assay is a reliable method for calibrating infectious titers of rcAAV reference standard materials, thereby enabling the standardization of rcAAV testing.