Immobilized Metal Affinity Adsorption for Antibody Depletion from Human Serum with Monosize Beads

Abstract

Iminodiacetic acid (IDA)-functionalized adsorbents have attracted increasing interest in recent years for immobilized metal-affinity chromatography (IMAC). In this study, IDA was covalently attached to nonporous monosize poly(glycidyl methacrylate) [poly(GMA)] beads (1.6 μm in diameter). Cu2+ ions were chelated via IDA groups for affinity depletion of immunoglobulin G (IgG) from human serum. The monosize poly(GMA) beads were characterized by scanning electron microscopy. The Cu2+-chelated beads (628 μmol/g) were used in the IgG adsorption−elution studies. Studies to determine the effects of IgG concentration, pH, and temperature on the adsorption efficiency of Cu2+-chelated beads were performed in a batch system. Nonspecific binding of IgG to monosize beads in the absence of Cu2+ ions was very low (0.45 mg/g). The IgG adsorption to chelated Cu2+ ions was 171.2 mg/g. The equilibrium IgG adsorption increased with increasing temperature. The negative change in Gibbs free energy (ΔGo < 0) indicated that the adsorption of IgG on the Cu2+-chelated beads was a thermodynamically favorable process. The ΔS and ΔH values were 172.1 J/mol·K and −43.2 kJ/mol, respectively. A significant amount of the adsorbed IgG (up to 97.2%) was eluted in the elution medium containing 1.0 M NaCl in 1 h. The kinetics of the interactions suggest that the interactions could be best represented by a mechanism based on second-order kinetics (k = 9.8 × 10-5 to 118.9 × 10-5 g·mg-1·min-1). The adsorption followed the Langmuir isotherm model with monolayer adsorption capacity of 156.2−212.8 mg/g. Consecutive adsorption−elution experiments showed that the Cu2+-chelated beads can be reused almost without any loss in the IgG adsorption capacity. To test the efficiency of IgG depletion from human serum, proteins in the serum and eluted portion were analyzed by two-dimensional gel electrophoresis. The depletion efficiency for IgG was above 98.2%. Eluted proteins include mainly IgG and a negligible amount of non-albumin proteins such as apo-lipoprotein A1, sero-transferrin, haptoglobulin, and α1-antitrypsin. When anti-HSA-Sepharose adsorbent is used together with our metal-chelated monosize poly(GMA) beads, IgG and HSA can be depleted in a single step.