A fundamental approach to buoyant density determination by DGE-AUC

Abstract

Density gradient equilibrium analytical ultracentrifugation (DGE-AUC) was first introduced in 1957. The method saw significant use over the following decade. Since then, DGE-AUC has been used by polymer and genomic DNA fields. Emerging medicine has revived interest in the technique for characterization of cell and gene therapeutics. While several model-dependent approaches exist to determine density at any point along a density gradient at equilibrium, there is ample evidence in the vast density gradient literature that indicates the presence of pressure effects, solvent compressibility, and general nonideal behavior of the gradient medium that are not easily accounted for in models describing the density gradient. These complications mandated the general use of reference materials and standard conditions. With an interest in buoyant density determination for particles of various composition, an approach that does not rely on standards is desirable. The current manuscript details a fundamental model-independent method for determination of buoyant density by DGE-AUC. An examination of this novel method is presented in the context of NISTmAb and DNA in a CsCl gradient, as well as polystyrene beads in a sucrose gradient. The method described herein is broadly applicable to determine the buoyant density of a particle in a density gradient medium.