Assessing DNA impurity integrity in rAAV gene therapy manufacturing
In this webinar, Dr. David Dobnik (National Institute of Biology) tackles a critical blind spot in gene therapy manufacturing: standard qPCR/dPCR impurity assays only detect small gene fragments, meaning they can't distinguish between harmless DNA debris and dangerous full-length, biologically active impurities—like intact antibiotic resistance genes or oncogenes—that recent studies show can enter human liver cells after AAV dosing.
Dr. Dobnik presents new multiplex digital PCR assays that solve this by using compartment-based linkage to confirm whether multiple gene targets co-occur on the same DNA molecule, revealing true sequence integrity rather than just fragment presence.Scientists working in viral vector characterization, CQA testing, or regulatory/safety assessment will want to watch for the full data and practical assay design details.
Key topics covered:
- Why fragment-based qPCR/dPCR testing may underestimate biosafety risk from full-length impurities
- How digital PCR compartments enable direct detection of sequence integrity via co-localization of multiple targets, without relying on Poisson statistics
- Challenges of multiplexing linkage calculations beyond duplex assays
- Case study results on recombinant AAV9 samples, including quantification of complete genes vs. fragmented/rearranged impurities
