After decades of
research, gene therapy products have
reached market
maturity in recent years. Recombinant adeno-associated viruses (rAAVs)
are one of the most promising gene delivery vehicles and are currently
under intense scientific investigation. These next-generation medicines
remain very challenging when it comes to designing appropriate analytical
techniques for quality control. One critical quality attribute is
the integrity of ssDNA incorporated in these vectors. The genome is
the active compound driving rAAV therapy and therefore requires proper
assessment and quality control. Current techniques for rAAV genome
characterization include next-generation sequencing, quantitative
polymerase chain reaction, analytical ultracentrifugation (AUC), and
capillary gel electrophoresis (CGE), yet each of them presents their
limitations or lack of user-friendliness. In this work, we demonstrate
for the first time the potential of ion pairing-reverse phase-liquid
chromatography (IP-RP-LC) to characterize the integrity of rAAV genomes.
The obtained results were supported by two orthogonal techniques,
AUC and CGE. IP-RP-LC can be performed above DNA melting temperatures,
avoiding the detection of secondary DNA isoforms, and does not require
the use of dyes due to UV detection. We demonstrate that this technique
is suitable for batch comparability, different rAAV serotypes (AAV2
and AAV8), internal vs external (inside vs outside the capsid) DNA
analysis, and contaminated samples. Overall, it is exceptionally user-friendly,
needs limited sample preparation, has high reproducibility, and permits
fractionation for further peak characterization. All of these factors
add significant value of IP-RP-LC to the analytical toolbox of rAAV
genome assessment.