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 LysB29 (Nɛω-carboxyheptadecanoyl) des(B30) human insulin and LysB29 (Nɛ-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 LysB29 (Nɛω-carboxyheptadecanoyl) des(B30) human insulin. Surprisingly, in the absence of zinc the two analogues behaved very differently. LysB29 (Nɛω-carboxyheptadecanoyl) des(B30) human insulin was in equilibrium between oligomers smaller than a hexamer, whereas LysB29 (Nɛ-lithocholyl) des(B30) human insulin self-associated and formed even larger complexes than in the presence of zinc.