After decades ofresearch, gene therapy products havereached marketmaturity in recent years. Recombinant adeno-associated viruses (rAAVs)are one of the most promising gene delivery vehicles and are... Show moreAfter decades ofresearch, gene therapy products havereached marketmaturity in recent years. Recombinant adeno-associated viruses (rAAVs)are one of the most promising gene delivery vehicles and are currentlyunder intense scientific investigation. These next-generation medicinesremain very challenging when it comes to designing appropriate analyticaltechniques for quality control. One critical quality attribute isthe integrity of ssDNA incorporated in these vectors. The genome isthe active compound driving rAAV therapy and therefore requires properassessment and quality control. Current techniques for rAAV genomecharacterization include next-generation sequencing, quantitativepolymerase chain reaction, analytical ultracentrifugation (AUC), andcapillary gel electrophoresis (CGE), yet each of them presents theirlimitations or lack of user-friendliness. In this work, we demonstratefor the first time the potential of ion pairing-reverse phase-liquidchromatography (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 requirethe use of dyes due to UV detection. We demonstrate that this techniqueis suitable for batch comparability, different rAAV serotypes (AAV2and AAV8), internal vs external (inside vs outside the capsid) DNAanalysis, and contaminated samples. Overall, it is exceptionally user-friendly,needs limited sample preparation, has high reproducibility, and permitsfractionation for further peak characterization. All of these factorsadd significant value of IP-RP-LC to the analytical toolbox of rAAVgenome assessment. Show less