Characterization of Human Serum Albumin Adsorbed on a Porous Anion-Exchange Support

Abstract

Fourier transform infrared (FTIR) spectroscopy and high-performance liquid chromatography (HPLC) were used to study the adsorption behavior of human serum albumin (HSA) onto a polymer-layer type anion exchanger, poly(vinylimidazole) (PVI) adsorbed and cross-linked on a porous silica support. The data are compared with previous results for HSA adsorbed on C6 alkyl chains grafted on the same silica matrix. For the adsorption onto the PVI support from a phosphate buffer solution of low ionic strength (pD 7.4), the FTIR experiments reveal only very weak structural and solvation changes. A large fraction of the protein remains irreversibly adsorbed and the amount retained at equilibrium is close to that observed for the adsorption on the reversed-phase support, although the structural effect of the stationary phase was much larger with the grafted C6 alkyl chains. Comparing to the solution state, only 2% of the HSA backbone is modified by adsorption on PVI, whereas 12% alterations are involved for the protein adsorbed on the reversed-phase support. When adsorbed from an eluent containing 20% acetonitrile, the amount of HSA retained by the PVI ion exchanger is about twice that measured with the buffer alone. This result is explained by a more compact structure of the protein when dissolved in the organoaqueous solvent. The presence of acetonitrile does not markedly affect the elution front of HSA adsorbed on the charged hydrophilic adsorbent. This result contrasts with the much lower apparent adsorption rate observed when HSA is adsorbed on the grafted alkyl chain support in the presence of acetonitrile in the buffer.