We describe the updated size-exclusion chromatography small angle X-ray scattering (SEC-SAXS) set-up used at the P12 bioSAXS beam line of the European Molecular Biology Laboratory (EMBL) at the PETRAIII synchrotron, DESY Hamburg (Germany). The addition of size exclusion chromatography (SEC) directly on-line to the SAXS capillary has become a well-established approach to reduce the effects of the sample heterogeneity on the SAXS measurements. The additional use of multi-angle laser light scattering (MALLS), UV absorption spectroscopy, refractive index (RI), and quasi-elastic light scattering (QELS) in parallel to the SAXS measurements enables independent molecular weight validation and hydrodynamic radius estimates. This allows one to address sample monodispersity as well as conformational heterogeneity. The benefits of the current SEC-SAXS set-up are demonstrated on a set of selected standard proteins. The processed SEC-SAXS data and models are provided in the Small Angle Scattering Biological Data Bank (SASBDB) and are labeled as “bench-marked” datasets that include the unsubtracted data frames spanning the respective SEC elution profiles and corresponding MALLS-UV-RI-QELS data. These entries provide method developers with datasets suitable for testing purposes, in addition to an educational resource for SAS data analysis and modeling.

We present a generically applicable approach to determine an extensive set of size-dependent critical quality attributes inside nanoparticulate pharmaceutical products. By coupling asymmetrical-flow field-flow fractionation (AF4) measurements directly in-line with solution small angle X-ray scattering (SAXS), vital information such as (i) quantitative, absolute size distribution profiles, (ii) drug loading, (iii) size-dependent internal structures, and (iv) quantitative information on free drug is obtained. Here the validity of the method was demonstrated by characterizing complex mRNA-based lipid nanoparticle products. The approach is particularly applicable to particles in the size range of 100 nm and below, which is highly relevant for pharmaceutical products—both biologics and nanoparticles. The method can be applied as well in other fields, including structural biology and environmental sciences.