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

Ryberg LA, Sønderby P, Barrientos F, Bukrinski JT, Peters GHJ, Harris P, Acta Crystallogr D Struct Biol 75(Pt 3):272-282 (2019) Europe PMC

SASDE26 – Albumin-insulin degludec 1:12 complex

Human Albumin (Recombumin(R) Elite, Albumedix Ltd.)
Insulin degludec(Tresiba(R), Novo Nordisk A/S)
MWexperimental 139 kDa
MWexpected 140 kDa
VPorod 179 nm3
log I(s) 1.02×10-1 1.02×10-2 1.02×10-3 1.02×10-4
Human Albumin (Recombumin(R) Elite, Albumedix Ltd.) Insulin degludec(Tresiba(R), Novo Nordisk A/S) small angle scattering data  s, nm-1
ln I(s)
Human Albumin (Recombumin(R) Elite, Albumedix Ltd.) Insulin degludec(Tresiba(R), Novo Nordisk A/S) Guinier plot ln 1.02×10-1 Rg: 3.8 nm 0 (3.8 nm)-2 s2
(sRg)2I(s)/I(0)
Human Albumin (Recombumin(R) Elite, Albumedix Ltd.) Insulin degludec(Tresiba(R), Novo Nordisk A/S) Kratky plot 1.104 0 √3 sRg
p(r)
Human Albumin (Recombumin(R) Elite, Albumedix Ltd.) Insulin degludec(Tresiba(R), Novo Nordisk A/S) pair distance distribution function Rg: 3.8 nm 0 Dmax: 13.4 nm

Data validation

Fits and models

log I(s)
 s, nm-1
Human Albumin (Recombumin(R) Elite, Albumedix Ltd.) Insulin degludec(Tresiba(R), Novo Nordisk A/S) SASREF CV model
log I(s)
 s, nm-1
Human Albumin (Recombumin(R) Elite, Albumedix Ltd.) Insulin degludec(Tresiba(R), Novo Nordisk A/S) DAMMIF model
Synchrotron SAXS data from solutions of Albumin-insulin degludec (1:12 complex) in 25 mM Na2HPO4, 15.9 mM m-cresol, 15.9 mM phenol, 212.8 mM glycerol, 20 mM NaCl, pH 7.6 were collected on the EMBL P12 beam line at the PETRA III storage ring (DESY, Hamburg, Germany) using a Pilatus 2M detector at a sample-detector distance of 3 m and at a wavelength of λ = 0.1241 nm (l(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). One solute concentration of 6.50 mg/ml was measured at 20°C. The data were normalized to the intensity of the transmitted beam and radially averaged; the scattering of the solvent-blank was subtracted.

SEC–SAXS with flow cell, effective sample path length 1.7 mm, flow rate 0.70 mL/min, 100 µL sample loaded with concentration 10.6 mg/mL. A Superdex 200 Increase 10/300 GL column (GE Healthcare Life Sciences) was used in combination with an Agilent 1260 Infinity Bio-Inert HPLC/FPLC machine Continuous 1 s SAXS data-frame measurements of SEC elution.

Human Albumin (Recombumin(R) Elite, Albumedix Ltd.)
Mol. type   Protein
Olig. state   Monomer
Mon. MW   66.5 kDa
 
UniProt   P02768 (25-609)
Sequence   FASTA
 
Insulin degludec(Tresiba(R), Novo Nordisk A/S)
Mol. type   Protein
Olig. state   Dodecamer
Mon. MW   6.1 kDa
 
UniProt   P01308 (25-110)
Sequence   FASTA
 
PDB ID   1BJ5
 
PDB ID   1TRZ