Characterisation of insulin analogues therapeutically available to patients

Abstract

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.

Solution structures of long-acting insulin analogues and their complexes with albumin

“…In agreement with previous studies (Steensgaard et al, 2013;Adams et al, 2018;Havelund et al, 2004), we find degludec as a dihexamer in phenol-containing buffer and detemir in a concentration-dependent equilibrium between hexamers, dihexamers, trihexamers and possibly larger multihexamers. We present the first structure of the detemir trihexamer, which has previously only been reported in a study using analytical ultracentrifugation (Adams et al, 2018). Surprisingly, the trihexamer has a bent shape.…”

“…Until recently, the highest oligomer of detemir reported was a dihexamer in equilibrium with a hexamer (Havelund et al, 2004), but in 2018 Adams and coworkers reported detemir in a trihexameric state in equilibrium with monomers, hexamers and dihexamers (Adams et al, 2018).…”

“…Whether the fatty-acid interactions are present in solution or simply an artefact induced by crystal packing is not clear (Whittingham et al, 1997). In solution, detemir has previously been shown to exist in an equilibrium between hexamers and dihexamers (Havelund et al, 2004), and a recent study using analytical ultracentrifugation sedimentation velocity showed that detemir is present in an equilibrium between monomers, hexamers, dihexamers and trihexamers (Adams et al, 2018).…”

“…The solution structure of degludec has been studied by both small-angle X-ray scattering (SAXS; Steensgaard et al, 2013) and analytical ultracentrifugation (Adams et al, 2017(Adams et al, , 2018Steensgaard et al, 2013). While there is general agreement that degludec is found as a dihexamer in phenol-containing solutions, different crystal forms show ambiguous intermolecular contacts (Steensgaard et al, 2013).…”

Analysis of insulin glulisine at the molecular level by X-ray crystallography and biophysical techniques

“…Although several characterisation studies have been published on IGlu 4 – 9 , its crystal structure has not been reported in detail. We have obtained crystals 10 and in this elucidation study, we identify for the first time, the results of crystallography combined with data from analytical ultracentrifugation to provide structural information that was previously lacking.…”