Development of a multi-detector asymmetric flow field-flow fractionation method for characterization of a protein nanoparticle antigen co-formulated with a squalene-based nanoemulsion adjuvant

Characterizing adjuvanted vaccine formulations can be challenging due to sample polydispersity, shear sensitivity, and potential secondary interactions with column stationary phases. Asymmetric flow field-flow fractionation (AF4) coupled with multiple downstream detectors offers a low-shear, size-based separation alternative to size-exclusion chromatography (SEC) to aid in vaccine development. In the present study, an AF4 method was developed to separate a protein nanoparticle antigen and a commercial squalene-based nanoemulsion adjuvant (AddaS03™). Method optimization included cross flow gradient screening, membrane selection and conditioning, dn/dc determination for AddaS03, and recovery assessment. The antigen eluted as a monodisperse peak (Rh ∼15 nm across entire peak, Mw ∼1.8 MDa, Mw/Mn ∼1.00), while AddaS03™ was highly polydisperse (Rh ∼ 66 nm, Mw ∼738 MDa) with Rg/Rh consistent with spherical morphology (0.77-1.02). Recoveries were 71-74% for AddaS03™ and >90% for the antigen. An exploratory thermal stress study induced formation of low-molecular-weight species and aggregation of the antigen. Observed changes in fractograms for co-formulated samples were attributable to the formation of a smaller population of adjuvant-associated particles and a ∼20% decrease in apparent weight-average molar mass. The new subpopulation of adjuvant particles overlaps in size with the antigen, which limits the component-specific data interpretation in stressed co-formulated samples. The optimized AF4 method enables simultaneous separation and characterization of intact protein nanoparticle antigens and nanoemulsion adjuvants within the same formulation, supporting stability studies while offering a low-shear environment.