Influence of the nature of coupling agents on insulin adsorption on supports grafted with sialic acid for high-performance affinity chromatography.
Porous silica exhibits excellent mechanical properties for use as a stationary phase for high-performance liquid chromatography. However, negative surface charges make it unusable in its native state. For this reason, silica beads are coated with dextran polymers carrying a calculated amount of diethylaminoethyl groups. Both the minimization of non-specific interactions and the hydrophilic character of such supports allow their functionalization with biospecific ligands and finally their use in high-performance affinity chromatography of biological products. The use of these modified supports in high-performance affinity chromatography requires a better understanding of various characteristics of stationary phases. For this purpose, several techniques were utilized, in particular, size-exclusion chromatography and adsorption of radiolabelled albumin. These methods provided complementary information on the structure of these supports. Coated silica-based supports were functionalized with sialic acid by means of different coupling agents. The affinity of these supports for insulin was determined by the establishment of adsorption isotherms and by high-performance affinity chromatography, to evidence the relationships between structural characteristics of the supports and their separation properties. The study of interactions between these supports and insulin allowed us to show the importance of the coupling method on the performances of supports in affinity chromatography.
Lakhiari
H
H
Laboratoire de Recherches sur les Macromolécules, CNRS URA 502, Université Paris Nord, Institut Galilée, Villetaneuse, France.
Jozefonvicz
J
J
Muller
D
D
D000327:Adsorption; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D007328:Insulin / Q000737:chemistry*; D007457:Iodine Radioisotopes; D007553:Isotope Labeling; D008863:Microspheres; D019158:N-Acetylneuraminic Acid; D012709:Serum Albumin / Q000737:chemistry; D012822:Silicon Dioxide
D007328:Insulin; D007457:Iodine Radioisotopes; D012709:Serum Albumin; D012822:Silicon Dioxide; D019158:N-Acetylneuraminic Acid