|Characterisation of insulin analogues with SEC-MALS and complementary ultracentrifugation
|BioPharma/Sample Prep & Automation
|Dr Richard Gillis
|Dr Shahwar Imran
Dr Gary Adams
|University of Nottingham
Abstract Information :
Insulin is a well-characterised, hormonal protein, fundamental to the homeostasis of blood-glucose levels. Diabetes is a condition where a patient is unable to maintain blood-glucose homeostasis, either through producing defective insulin (type 1) or not producing enough to elicit a body-wide response (type 2).
Along with a range of other interventions, a common treatment method is for regular subcutaneous injections of insulin. Patients and clinicians have access to a wide choice of insulin formulations, including analogues (sometimes referred to as biosimilars). These insulin analogues have been modified either in their amino acid sequence or by conjugation. Some modifications yield a faster pharmacokinetic response (FASTS: aspart, glulisine, lispro) and some yield a slower, basal response (SLOWS: detemir, degludec, glargine (not assessed)). Studying the properties of these analogues is critical to understanding their functions in the body.
Size Exclusion Chromatography coupled to Multi-Angle Light Scattering (SEC-MALS) was employed to investigate the properties of these analogues. Analytical ultracentrifugation (AUC) was also used as a complementary technique. SEC-MALS used PBS, Tris and nitrate mobile phases at pH7 and small quantities of cyclohexanol. Insulins were injected as their off-the-shelf formulations, including small quantities of phenol, m-cresol and zinc, although exact concentrations varied between samples/suppliers. AUC used just the off-the-shelf formulations.
SEC-MALS revealed that ‘NATIVE’ insulins (human recombinant, bovine, porcine) eluted as a single peak, with light scattering confirming the presence of a hexameric stoichiometry. Fast-acting analogues eluted as broad monomer-dimer systems. Slow-acting analogues eluted as dihexamers. AUC, however, suggested that NATIVES and FASTS exist mostly as the hexamer, except for glulisine which sedimented as a complex dimer-hexamer-dihexamer system. SLOWS, however, were consistent with SEC-MALS.
These results are consistent with the mode of actions of these analogues – FASTS dissociate faster from the hexamer than NATIVES, whereas SLOWS are more likely to self-associate. This is most likely due to the contribution of zinc ions, which are known to stabilise the hexamer. Insulin is most effective as a monomer and stays inactive as a hexamer, therefore FASTS are more likely to dissociate and act quicker than NATIVES. SLOWS remain as complex poly-hexamers for a long time, therefore protracting their mode of action for up to 48 hours.