Affinity Membranes:  Competitive Binding of the Human IgG Subclasses to Immobilized Protein G

Abstract

Sorption of the four subclasses of human immunoglobulin G (hIgG) to recombinant protein G immobilized to microporous membranes was examined to further the understanding and characterization of this medicallyimportant system. Using batch incubation, sorption of the individual hIgG subclasses was measured in competitive and noncompetitive experiments. Individually, all subclasses had very similar sorption rates and equilibrium capacities. In contrast, for mixtures of the subclasses, binding was distinctly different, with strong competitive binding occurring: as the system approached equilibrium, significantly more hIgG1 and hIgG3 bound to the membrane than did hIgG2 and hIgG4. A kinetic and equilibrium model was able to successfully simulate the binding of hIgG1, hIgG2, and hIgG4 but not hIgG3. The results are relevant to the healthcare and biotechnology industries: (1) the diagnosis and treatment of autoimmune diseases, (2) mitigation of rejection in recipients of organ transplants, and (3) production of monoclonal antibodies for use as therapeutic biopharmaceuticals.

Feasibility Study for the Fractionation of the Major Human Immunoglobulin G Subclasses Using Hydrophobic Interaction Membrane Chromatography

“…Earlier workers have shown that these techniques discriminate between the different subclasses. 13,16 The subclass composition of human IgG used in our study could, therefore, be significantly different from that in whole serum. Using appropriate calibration, the HIMC technique could be used to determine relative and absolute IgG1 and IgG2 content in a human serum.…”

“…11,12 In a more recent study, the competitive binding of the different subclasses on protein-G has been discussed. 13 However, the differences in binding were not significant enough to be exploited for subclass fractionation. Moreover, separation based on the differential binding on protein-A or protein-G would involve multiple steps with pH adjustments being required in between.…”

“…Protein-A based affinity chromatography has been found to be suitable for isolating IgG3 from whole IgG. , In a more recent study, the competitive binding of the different subclasses on protein-G has been discussed . However, the differences in binding were not significant enough to be exploited for subclass fractionation.…”

Oriented Immobilization of Antibodies with GST-Fused Multiple Fc-Specific B-Domains on a Gold Surface

“…The present immunoarray system appears to be applicable to a selection method for monoclonal antibodies, a detection of protein expression, or a diagnostics for a disease by means of analyzing a serum sample from a patient. Although plenty of IgG in a serum sample (in the concentration of about several mg/mL) will eventually replace the diagnostic antibodies immobilized on GST-GB 3 after a long incubation time, the dissociation constant ( k d , ∼10 -5 s -1 ) of an immunoglobulin from protein G seems to be substantially low enough to retain the structural integrity of antibody/GST-GB 3 systems from a 1 or 2 h soaking in a serum sample . In addition, no noticeable dissociation in a SPR sensorgram is observed within 2 h, when a blank buffer solution is allowed to the SPR channel functionalized with an IgG/GST-GB 3 layer.…”

Purification of a Large Protein Using Ion-Exchange Membranes

“…Membrane chromatography is a relatively new purification technology designed to bypass the fundamental limitations of columns packed with beads. − In membrane chromatography, the packing consists of microporous membranes wherein the internal pores contain adsorptive moieties that bind the target protein. Because the membranes are thin, pressure drop is not a limitation.…”