Self-association of long-acting insulin analogues studied by size exclusion chromatography coupled to multi-angle light scattering

TY - JOUR

T1 - Self-association of long-acting insulin analogues studied by size exclusion chromatography coupled to multi-angle light scattering

AU - Jensen, Malene Hillerup

AU - Wahlund, Per-Olof

AU - Jacobsen, Jes K.

AU - Vestergaard, Bente

AU - van de Weert, Marco

AU - Havelund, Svend

PY - 2011/10/15

Y1 - 2011/10/15

N2 - Two structurally very different insulin analogues analysed here, belong to a class of analogues of which two have been reported to have a protracted action through self-assembly to high molar mass in subcutis. The process of self-association of insulin analogues Lys B29 (N e{open}ω-carboxyheptadecanoyl) des(B30) human insulin and Lys B29 (N e{open}-lithocholyl) des(B30) human insulin was investigated using size exclusion chromatography (SEC) in connection with multi-angle light-scattering. Self-assembly to high molar mass was obtained by exchanging the formulation containing phenolic preservatives with an isotonic eluent during SEC. It was shown that increasing amounts of zinc in the formulations of the two analogues increased the size of the self assemblies formed during gel filtration. The addition of 0.2mM phenol to the elution buffer slowed down the self-association process of zinc containing formulations and shed light on the initial association process. The results indicated that a dihexamer is a possible building block during self-association of Lys B29 (N e{open}ω-carboxyheptadecanoyl) des(B30) human insulin. Surprisingly, in the absence of zinc the two analogues behaved very differently. Lys B29 (N e{open}ω-carboxyheptadecanoyl) des(B30) human insulin was in equilibrium between oligomers smaller than a hexamer, whereas Lys B29 (N e{open}-lithocholyl) des(B30) human insulin self-associated and formed even larger complexes than in the presence of zinc.

AB - Two structurally very different insulin analogues analysed here, belong to a class of analogues of which two have been reported to have a protracted action through self-assembly to high molar mass in subcutis. The process of self-association of insulin analogues Lys B29 (N e{open}ω-carboxyheptadecanoyl) des(B30) human insulin and Lys B29 (N e{open}-lithocholyl) des(B30) human insulin was investigated using size exclusion chromatography (SEC) in connection with multi-angle light-scattering. Self-assembly to high molar mass was obtained by exchanging the formulation containing phenolic preservatives with an isotonic eluent during SEC. It was shown that increasing amounts of zinc in the formulations of the two analogues increased the size of the self assemblies formed during gel filtration. The addition of 0.2mM phenol to the elution buffer slowed down the self-association process of zinc containing formulations and shed light on the initial association process. The results indicated that a dihexamer is a possible building block during self-association of Lys B29 (N e{open}ω-carboxyheptadecanoyl) des(B30) human insulin. Surprisingly, in the absence of zinc the two analogues behaved very differently. Lys B29 (N e{open}ω-carboxyheptadecanoyl) des(B30) human insulin was in equilibrium between oligomers smaller than a hexamer, whereas Lys B29 (N e{open}-lithocholyl) des(B30) human insulin self-associated and formed even larger complexes than in the presence of zinc.

KW - Former Faculty of Pharmaceutical Sciences

U2 - 10.1016/j.jchromb.2011.05.059

DO - 10.1016/j.jchromb.2011.05.059

M3 - Journal article

SN - 1570-0232

VL - 879

SP - 2945

EP - 2951

JO - Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences

JF - Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences

IS - 28

ER -