Characterisation of insulin analogues therapeutically available to patients

The structure and function of clinical dosage insulin and its analogues were assessed. This included ‘native insulins’ (human recombinant, bovine, porcine), ‘fast-acting analogues’ (aspart, glulisine, lispro) and ‘slow-acting analogues’ (glargine, detemir, degludec). Analytical ultracentrifugation, both sedimentation velocity and equilibrium experiments, were employed to yield distributions of both molar mass and sedimentation coefficient of all nine insulins. Size exclusion chromatography, coupled to multi-angle light scattering, was also used to explore the function of these analogues. On ultracentrifugation analysis, the insulins under investigation were found to be in numerous conformational states, however the majority of insulins were present in a primarily hexameric conformation. This was true for all native insulins and two fast-acting analogues. However, glargine was present as a dimer, detemir was a multi-hexameric system, degludec was a dodecamer (di-hexamer) and glulisine was present as a dimer-hexamer-dihexamer system. However, size-exclusion chromatography showed that the two hexameric fast-acting analogues (aspart and lispro) dissociated into monomers and dimers due to the lack of zinc in the mobile phase. This comprehensive study is the first time all nine insulins have been characterised in this way, the first time that insulin detemir have been studied using analytical ultracentrifugation and the first time that insulins aspart and glulisine have been studied using sedimentation equilibrium. The structure and function of these clinically administered insulins is of critical importance and this research adds novel data to an otherwise complex functional physiological protein.

本研究对临床使用剂量的胰岛素及其类似物的结构与功能进行了评估，涵盖“天然胰岛素（native insulins）”（包括人重组胰岛素、牛胰岛素、猪胰岛素）、“速效类似物（fast-acting analogues）”（门冬胰岛素、谷赖胰岛素、赖脯胰岛素）以及“长效类似物（slow-acting analogues）”（甘精胰岛素、地特胰岛素、德谷胰岛素）。本研究采用分析超速离心技术（analytical ultracentrifugation），包含沉降速度与沉降平衡实验，测定了全部9种胰岛素的摩尔质量与沉降系数分布；同时采用多角度光散射联用尺寸排阻色谱法（size exclusion chromatography coupled to multi-angle light scattering）探究了这些胰岛素类似物的功能特性。

通过超速离心分析发现，受试胰岛素存在多种构象状态，但绝大多数胰岛素主要以六聚体构象存在，所有天然胰岛素与两种速效类似物均符合这一特征。不过，甘精胰岛素以二聚体形式存在，地特胰岛素为多六聚体体系，德谷胰岛素为十二聚体（双六聚体），而谷赖胰岛素则以二聚体-六聚体-双六聚体混合体系形式存在。但尺寸排阻色谱分析结果显示，由于流动相缺乏锌离子，两种呈六聚体状态的速效类似物（门冬胰岛素与赖脯胰岛素）解离为单体与二聚体。

这项综合性研究首次采用上述表征方法对全部9种胰岛素进行了系统性分析；首次通过分析超速离心技术对地特胰岛素展开研究，同时首次利用沉降平衡实验对门冬胰岛素与谷赖胰岛素进行表征。临床使用的胰岛素的结构与功能至关重要，本研究为这类结构复杂且具备重要生理功能的蛋白质补充了全新的实验数据。