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International audience; Nanoformulations are complex systems where physicochemical properties determine their therapeutic efficacy and safety. In the case of nanovaccines, particle size and shape play a crucial role on the immune response generated. Furthermore, the antigen's integrity is also a key aspect to control when producing a nanovaccine. The determination of all those physicochemical properties is still an analytical challenge and the lack of well-established methods hinders the access of new therapeutics to the market. In this work, robust methods for the characterization of a novel HIV nanoparticle-based vaccine produced in good manufacturing practice (GMPs)-like environment were developed. With slightly polydisperse particles (< 0.2) close to 180 nm of size, batch-mode Dynamic Light Scattering (DLS) was validated to be used as a quality control technique in the pilot production plant. In addition, a high size resolution method using Asymmetrical Flow Field Flow Fractionation (AF4) demonstrated its ability to determine not only size and size distribution but also shape modification across the size and accurate quantification of the free active ingredient. Results showed a monomodal distribution of particles from 60 to 700 nm, most of them (> 90%) with size lower than 250 nm, consistent with more traditional techniques, and revealed a slight change in the structure of the particles induced by the presence of the antigen. Finally, a batch to batch variability lower than 20% was obtained by both DLS and AF4 methods indicating that preparation method was highly reproducible.