Abstract
Gene therapy using adeno-associated virus (AAV) vectors has emerged as a promising strategy, offering new hope for patients with genetic disorders. However, during rAAV manufacturing, residual host-cell DNA (hcDNA) and plasmid DNA from the production process may remain in the final product. These impurities are classified as Critical Quality Attributes (CQAs) for release testing, and accurate quantification of these contaminants is essential to mitigate potential safety risks and ensure regulatory compliance. To address this requirement, a droplet digital PCR (ddPCR) method was developed and validated in accordance with ICH Q2(R1) guidelines for the quantification of residual E1A gene sequence, a viral oncogene constitutively expressed in Pro10™ and other HEK293-derived producer cell lines, which plays a critical role in rAAV production.
For the E1A-ddPCR method, rAAV samples were pretreated with and without DNase I, DNA was extracted using a semi-automated magnetic beads-based system and analyzed by ddPCR targeting several E1A amplicons of different lengths. This design enabled quantification of both total residual E1A DNA (free, encapsidated, or capsid-associated) and encapsidated and/or capsid-associated residual E1A DNA (DNase-resistant), as well as fragmentation analysis to assess DNA integrity.
The validated method demonstrated adequate repeatability (CV ≤ 3%), intermediate precision (CV ≤ 5%), and accuracy (83 - 100%) across a defined range using the QX200 and QX ONE ddPCR systems. Specificity against the target sequence or other components and/or impurities regularly found in the test samples was also confirmed, and robustness testing ensured reliability under variation of key parameters: different primers/probe lots, plate storage, DNase activity, and the use of QX200 vs. QX ONE system. Additionally, correlation analysis with total hcDNA levels in rAAV samples suggested similar behavior of total hcDNA based on 18S and E1A sequences during encapsidation and purification.
Overall, this analytical approach for impurity detection strengthens quality control for product characterization and process development.
Get the full details in our scientific poster: ddPCR-Based Quantification and Fragment Analysis of Residual E1A in rAAV Products: Method Validation and Correlation with Total Host Cell DNA
