Fig 1.
Primary structure of human insulin and its analogues.
Differences highlighted and numbered.
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.
Table 1.
Hydrodynamic parameters measured using AUC-SV and AUC-SE.
Sedimentation coefficients are weight-averaged in the cases of multi-species distributions. IGlu and IDet yielded fits with BM<0. All insulins were measured at ~3.5mg/mL, except for IDet which was measured at 14.2mg/mL.
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.
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.
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.
Table 2.
Weight-average molar mass estimates from AUC-SV, AUC-SE and SEC-MALS results.
Estimates uncorrected for non-ideality are labelled ‘apparent’ (app). All AUC-SV estimates were made on data from 45k rpm and based upon estimates of the frictional ratio fitted from boundary spread, and are weight averaged in the case of multiple sedimenting species. AUC-SE analysis was made at the indicated rotor speeds in parentheses (k rpm). SEC-MALS estimates are shown as consensus values from PBS and TRIS mobile phases. IDeg was unanalysable using MSTAR and MULTISIG. IGla was not assayed using SEC-MALS due to pI/pH incompatibilities.