Self-association of long-acting insulin analogues studied by size exclusion chromatography coupled to multi-angle light scattering.

Two structurally very different insulin analogues analysed here, belong to a class of analogues of which two have been reported to have a protracted action through self-assembly to high molar mass in subcutis. The process of self-association of insulin analogues Lys(B29) (N(ε)ω-carboxyheptadecanoyl) des(B30) human insulin and Lys(B29) (N(ε)-lithocholyl) des(B30) human insulin was investigated using size exclusion chromatography (SEC) in connection with multi-angle light-scattering. Self-assembly to high molar mass was obtained by exchanging the formulation containing phenolic preservatives with an isotonic eluent during SEC. It was shown that increasing amounts of zinc in the formulations of the two analogues increased the size of the self assemblies formed during gel filtration. The addition of 0.2 mM phenol to the elution buffer slowed down the self-association process of zinc containing formulations and shed light on the initial association process. The results indicated that a dihexamer is a possible building block during self-association of Lys(B29) (N(ε)ω-carboxyheptadecanoyl) des(B30) human insulin. Surprisingly, in the absence of zinc the two analogues behaved very differently. Lys(B29) (N(ε)ω-carboxyheptadecanoyl) des(B30) human insulin was in equilibrium between oligomers smaller than a hexamer, whereas Lys(B29) (N(ε)-lithocholyl) des(B30) human insulin self-associated and formed even larger complexes than in the presence of zinc.

High-throughput small angle X-ray scattering from proteins in solution using a microfluidic front-end.

This manuscript presents, for the first time, the method of automated structural analysis of biomolecules in solution on a microfluidic chip. A polymer-based micrototal analysis system for high-throughput Small-Angle X-ray Scattering (SAXS) data collection from biological macromolecules has been developed. The bioXTAS chip features an integrated X-ray transparent 200 nL sample chamber and diffusion-based mixing of protein and buffer solutions. Software for fully automated fluidic control, data acquisition, and data analysis has been developed. The proof-of concept is based on data using bovine serum albumin as the model system. It confirms the quality of SAXS data generated from small sample volumes and furthermore validates the on-chip mixing capabilities. SAXS data on the gradual unfolding of BSA induced by an anionic surfactant exemplifies how the bioXTAS chip can be used to follow and identify structural changes and proves the feasibility of high-throughput structural analysis in solution. In total, this shows that the bioXTAS chip has the potential for becoming a powerful tool for automated high-throughput structural analysis of macromolecular systems.