Affinity separation with polyaldehyde microsphere beads

Agarose polyaldehyde microsphere beads were prepared by encapsulating polyaldehyde microspheres of various diameters, e.g., polyacrolein or polyglutaraldehyde microspheres, within agarose beads. Amino ligands such as proteins or drugs can be bound covalently to the beads in a single step at physiological pH. The binding capacity of the beads towards various amino ligands is inversely related to the diameter of the microspheres encapsulated in the agarose matrix. Different reagents, e.g., bovine serum albumin, ethanolamine and hydroxylamine, were studied as blocking reagents of the free aldehyde groups. Blocking the remaining aldehyde groups after coupling the amino ligands to the beads is essential for increasing or retaining the reactivity of the ligands conjugated to the beads. Among the reagent studied, hydroxylamine was found to be the most suitable blocking reagent of the free aldehyde groups of beads conjugated with proteins. The extent of leakage of amino ligands bound to the agarose-polyaldehyde microsphere beads was studied as a function of the pH of aqueous solutions of the beads. At physiological pH the leakage was negligible. At acid pH, leakage of ligands containing several primary amine groups, e.g., proteins, was insignificant. However, significant leakage was detected for ligands containing a single amino group. The leakage of proteins bound to the agarose-polyaldehyde microsphere beads was found to be much less than the leakage of the same proteins bound to agarose beads through the cyanogen bromide activation method.