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. 2018 Mar 29;13(3):e0195010.
doi: 10.1371/journal.pone.0195010. eCollection 2018.

Characterisation of insulin analogues therapeutically available to patients

Gary G Adams  1   2 , Andrew Meal  1 , Paul S Morgan  1 , Qushmua E Alzahrani  1   2   3 , Hanne Zobel  4 , Ryan Lithgo  2 , M Samil Kok  1   5 , David T M Besong  6 , Shahwar I Jiwani  1   2 , Simon Ballance  4 , Stephen E Harding  2 , Naomi Chayen  7 , Richard B Gillis  1   2
Affiliations

Characterisation of insulin analogues therapeutically available to patients

Gary G Adams et al. PLoS One. .

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.

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Conflict of interest statement

Competing Interests: Affiliations between GA and the Independent Diabetes Trust, and HZ, SB and Nofima AS, do not alter our adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1
Fig 1. Primary structure of human insulin and its analogues.
Differences highlighted and numbered.
Fig 2
Fig 2. c(s) vs. sedimentation coefficient distributions measured using AUC-SV at 45k rpm.
(a) Native insulins; (b) Rapid-acting analogues; (c) Slow-acting analogues. Monomers (M), Dimers (D), Hexamers (H), and Di and Tri Hexamers (Di/TriH) were identified by molar mass calculated through the sedimentation coefficient and the frictional ratio.
Fig 3
Fig 3. c(M) vs. molar mass distributions measured using SEDFIT-MSTAR from AUC-SE data, with superimposed M*(r) extrapolations to the cell base (relative radius = 1), and c(M) fits over raw AUC-SE data in insets.
(a) IHr; (b) IBov; (c) IPor; (d) IAsp; (e) IGlu; (f) ILis; (g) IGla; (h) IDet; (i) IDeg. SEDFIT-MSTAR was unable to adequately fit data from IDeg.
Fig 4
Fig 4. Molar mass distributions measured against concentration using MULTISIG-RADIUS from AUC-SE data, with superimposed n-, w- and z-average molar masses.
Insets are fits from INVEQ analysis. (a) IHr; (b) IBov; (c) IPor; (d) IAsp; (e) IGlu; (f) ILis; (g) IGla; (h) IDet; (i) IDeg. MULTISIG-RADIUS was unable to adequately fit data from IDeg, but INVEQ was able.
Fig 5
Fig 5. Elution from SEC plots of insulin and analogues.
Black line represents PBS as the mobile phase, grey line represents TRIS. (a) IHr; (b) IBov; (c) IPor; (d) IAsp; (e) IGlu; (f) ILis; (g) IDet; (h) IDeg. IGla was not injected due to pI/pH incompatibilities. Monomers (M), Dimers (D), Hexamers (H), Dihexamers (DiH), multihexamers (multiH) and Excipients (Ex) were identified by molar mass.
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