An Improved Method for Determining the Infection Titer of Replication-Competent Adeno-Associated Virus
Brief intro:
- Author: Jianning Fu, Lei Yu, Zhihao Fu, Guangyu Wang, Chenggang Liang, Xinchang Shi, Yixuan Zhang
- Journal: Biomedicines
- Doi: https://www.doi.org/10.3390/biomedicines14030653
- Publication Date: 2026/3/13
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.
About PackGene
PackGene Biotech is a world-leading CRO and CDMO, excelling in AAV vectors, mRNA, plasmid DNA, and lentiviral vector solutions. Our comprehensive offerings span from vector design and construction to AAV, lentivirus, and mRNA services. With a sharp focus on early-stage drug discovery, preclinical development, and cell and gene therapy trials, we deliver cost-effective, dependable, and scalable production solutions. Leveraging our groundbreaking π-alpha 293 AAV high-yield platform, we amplify AAV production by up to 10-fold, yielding up to 1e+17vg per batch to meet diverse commercial and clinical project needs. Moreover, our tailored mRNA and LNP products and services cater to every stage of drug and vaccine development, from research to GMP production, providing a seamless, end-to-end solution.
