Adenoviral vectors have been widely used in gene therapy clinical trials and subjected to rigorous testing to ensure safety and efficacy. Like therapeutic proteins, aggregation of adenoviral vectors needs to be quantified for process consistency and stability monitoring. The sucrose gradient sedimentation method of adenovirus particles using disc centrifugation, which is a modification of a method described by Bondoc and Fitzpatrick, was used. It proved to be quantitative and reproducible in evaluating a variety of samples including deliberately cross-linked adenovirus particles and process development lots of various ages. This aggregation assay revealed that most aggregates detected in the production lots were dimers, trimers, and tetramers; and the number of these small oligomers was easily reduced with the addition of 300 mM salt, thus demonstrating the reversible nature of a portion of the aggregate population. This method was validated to demonstrate that it was appropriate for final product lot release and stability monitoring…