Journal of Chromatography A, 989 (2003) 131–138 www.elsevier.com / locate / chroma Interaction of immunoglobulin G with N,N,N9,N9- ethylenediaminetetramethylenephosphonic acid-modified zirconia a, b * Anuradha Subramanian , Sabyasachi Sarkar a Department of Chemical Engineering, University of Nebraska, Lincoln, NE 68588, USA b Biosystems and Agricultural Engineering, University of Minnesota, St. Paul, MN 55108, USA Abstract Zirconia beads (25–38 mm in diameter) were modified with N,N,N9,N9-ethylenediaminetetramethylenephosphonic acid to generate a pseudo-biospecific support, r PEZ. To better understand the force of interaction between the IgG and the r PEZ, ] ] the equilibrium dissociation constant ( K ) was determined by static binding isotherms, as a function of temperature and by d frontal analysis at different linear velocities. Temperature had no significant impact on the maximum static binding capacity ( Q ) and the equilibrium-binding constant ( K ), whereas pH and the salt concentration had a noticeable impact on both max d Q and K values. Q was found to be in the range of 55–65 mg IgG per ml of beads and unaffected by temperature. max d max The maximum dynamic binding capacity (Q ) was found to be in the range of 20–12 mg IgG per ml of beads. The x 21 21 adsorption rate constant (k ) was determined by a split-peak approach to be between 982 and 3242 l mol s depending on a the linear velocity. Adsorption rate of IgG on r PEZ was studied as a function of both feed concentration and linear velocity. ] The standard enthalpy and entropy values were estimated for the interaction of IgG with this novel support. The binding constants were also determined by modeling the batch protein-uptake data.  2002 Elsevier Science B.V. All rights reserved. Keywords: Zirconia; Adsorption; Thermodynamic parameters; Binding studies; Immunoglobulins; Proteins; Ethyl- enediaminetetramethylenephosphonic acid 1. Introduction scalable protein separation methodologies. Research- based prediction of mass transport, biological activi- Economics, efficiency and practicality are some of ty behavior, kinetic and thermodynamic parameters the constraints dictating the search for novel chro- that impact protein retention and separation are thus matographic supports relevant for use in the in- essential for the integration of these chromatograph- dustrial or large-scale purification of proteins. In this ic-based unit-operations into the purification scheme. regard, affinity chromatography, ion-exchange chro- Affinity chromatography uses biological ligands matography and other forms of electrostatic chroma- like protein A or biotin–avidin to achieve exquisite tography that exploit differences in biological spe- specificity and separation. However, many of these cificity or surface charge anisotropy of proteins, have biological ligands are macromolecular and fragile, the greatest potential of impacting future trends in expensive to obtain from bacterial or tissue-culture sources and are difficult to immobilize without losing activity. The use of protein A and protein G in *Corresponding author. Tel.: 11-402-472-3463; fax: 11-402- affinity chromatography is also negatively impacted 472-6989. E-mail address: asubramanian2@unl.edu (A. Subramanian). by the harsh elution conditions and pose special 0021-9673 / 02 / $ – see front matter  2002 Elsevier Science B.V. All rights reserved. PII: S0021-9673(02)01901-5