Stability of Asp(B28) Insulin Exposed to Modified and Unmodified Polypropylene.

Polypropylene (PP) plates have been modified with two different hydrophilic polymeric materials, poly(N,N-dimethylacrylamide) (poly(DMAAm)) and poly(poly(ethylene glycol)methacrylate) (poly(PEGMA)) in order to reduce insulin adsorption when the plates were exposed to insulin aspart (Asp(B28) insulin). The influence of surface modification on the chemical and physical stability of Asp(B28) insulin was evaluated by two chromatographic methods, size exclusion chromatography (SEC) and reverse phase high pressure liquid chromatography (RP-HPLC) and the Thioflavin T assay. A clear difference in the stability of Asp(B28) insulin was observed between the three tested surfaces. PP coated with poly(DMAAm) resulted in a poor chemical stability and a significantly improved physical stability compared with unmodified PP. In addition to this a lower phenol concentration was observed for the poly(DMAAm) coating. The results from the poly(PEGMA) coating looked very promising with respect to the stability of Asp(B28) insulin in comparison with the data from unmodified PP and the poly(DMAAm) coating. Two hydrophilic coatings have been tested and surprisingly a difference in Asp(B28) insulin stability was observed. Therefore, Asp(B28) insulin adsorption and stability will be influenced by more than the hydrophilicity of the surface.

Influence of acylation on the adsorption of GLP-2 to hydrophobic surfaces.

Acylation of proteins with a fatty acid chain has proven useful for prolonging the plasma half-lives of proteins. In formulation of acylated protein drugs, knowledge about the effect of acylation with fatty acids on the adsorption behaviour of proteins at interfaces will be valuable. The aim of this work was to study the effect of acylation on the adsorption of GLP-2 from aqueous solution to a hydrophobic surface by comparing the adsorption of the 3766 Da GLP-2 with that of a GLP-2 variant acylated with a 16-carbon fatty acid chain through a ß-alanine linker. Adsorption of GLP-2 and acylated GLP-2 were studied with isothermal titration calorimetry, fixed-angle optical reflectometry and total internal reflection fluorescence. Furthermore, the effect of acylation of GLP-2 on the secondary structure was studied with Far-UV CD. Acylation was observed to have several effects on the adsorption of GLP-2. Acylation increased the amount of GLP-2 adsorbing per unit surface area and decreased the initial adsorption rate of GLP-2. Finally, acylation increased the strength of the adsorption, as judged by the lower fraction desorbing upon rinsing with buffer.

Adsorption of insulin with varying self-association profiles to a solid Teflon surface--influence on protein structure, fibrillation tendency and thermal stability.

Interfaces are present in the preparation of pharmaceutical products and are well known for having an influence on the physical stability of proteins. The aim of this study was to examine the conformation (i.e. secondary and tertiary structures) and fibrillation tendency, overall aggregation tendency and thermal stability of adsorbed human insulin at a solid particulate Teflon surface. The effects of changes in the association degree of insulin on the structure and stability have been determined. Using SEC-HPLC, association profiles were determined for insulin aspart, zinc-free human insulin and human insulin with two Zn(2+) per hexamer in concentrations ranging from 0.1 mg/ml to 20 mg/ml. Insulin aspart was 100% monomeric, regardless of concentration. In contrast, human insulin went from 100% monomer to 80% hexamer, and 20% dimer/monomer and zinc-free human insulin from 100% monomer to 70% dimer and 30% monomer with increasing concentration. The secondary structure of the insulins changed upon adsorption, but only minor differences were observed among the insulins. Structural changes were observed when the insulin-surface ratio was varied, but at no point did the structure resemble that of fibrillated insulin in solution. The presence of particles resulted in increased fibrillation of human insulin. The lag-time of fibrillation decreased, when the amount of particles present was increased. In conclusion, the type and association degree of the three insulin variants has no major influence on the secondary structure observed after adsorption of insulin at the solid Teflon surface. However, the presence of particles increases the tendency of insulin to fibrillate.

Binding mode of Thioflavin T in insulin amyloid fibrils.

Amyloid fibrils share various common structural features and their presence can be detected by Thioflavin T (ThT). In this paper, the binding mode of ThT to insulin amyloid fibrils was examined. Scatchard analysis and isothermal titration calorimetry (ITC) showed at least two binding site populations. The binding site population with the strongest binding was responsible for the characteristic ThT fluorescence. This binding had a capacity of about 0.1 moles of ThT bound per mole of insulin in fibril form. The binding capacity was unaffected by pH, but the affinity was lowest at low pH. Notably, presence of a third binding process prior to the other processes was suggested by ITC. Binding of ThT resulted in only minor changes in the fibril structure according to the X-ray diffraction patterns, where a slightly more dominant equatorial reflection at 16A relative to the intersheet distance of 11A was observed. No change in the interstrand distance of 4.8A was observed. On the basis of our results, we propose that ThT binds in cavities running parallel to the fibril axis, e.g., between the protofilaments forming the fibrils. Such cavities have been proposed previously in insulin fibrils and several other amyloid fibril models.

Interfacial adsorption of insulin conformational changes and reversibility of adsorption.

The adsorption of human insulin to Teflon particles was studied with respect to conformational changes and the reversibility of adsorption was examined by total internal reflection fluorescence (TIRF). Adsorption isotherms for the adsorption of human insulin indicated high affinity adsorption, even at electrostatic repulsive conditions. The plateau value for adsorption was in accordance with a protein layer consisting primarily of insulin monomers. Conformational changes of the insulin upon adsorption, was investigated by circular dicroism (CD) and fluorescence spectroscopy. The results suggested unfolding of adsorbed insulin, as observed by a decrease in alpha-helix and increase in random coil conformation. The changes in protein structure was not only related to the adsorbed species being monomeric, since CD and fluorescence results were different for adsorbed insulin compared to a monomeric analog of human insulin. Furthermore, the thermal stability in the adsorbed state was changed compared to insulin in solution. On the basis of the TIRF studies with FITC-labelled insulin it was not possible to firmly conclude whether exchange between human insulin in the adsorbed state and in solution takes place, due to the limited time range investigated. However, the desorption mechanism appeared to be different with unlabelled insulin in the bulk solution compared to phosphate buffer.

Complex of sialoadhesin with a glycopeptide ligand.

Sialoadhesin is a sialic acid-binding immunoglobulin-like lectin (Siglec), expressed on subsets of macrophages. It is a model system for Siglec receptor-mediated cell surface interactions through binding of sialylated glycoconjugates. The N-terminal sialoadhesin domain can mediate sialic acid-binding on its own. The structure of this domain has been determined in complex with a sialic acid-containing heptapeptide, (Ala-Gly-His-Thr(Neu5Ac)-Trp-Gly-His). The affinity of sialoadhesin for this ligand is four times higher than the affinity for the natural linkage 2,3'-sialyllactose. The structure of the glycopeptide complex suggests strategies for ligand optimization and provides possible explanations for the observed differences in specificities among the Siglecs.