title: Development of L-histidine immobilized CIM(®) monolithic disks for purification of immunoglobulin G.
abstract: The pseudobiospecific affinity ligand l-histidine was immobilized on epoxy, carbonyldiimidazole (CDI), and ethylenediamine (EDA) convective interaction media (BIA Separations, Slovenia) monolithic disks to obtain the histidyl affinity column for purification of immunoglobulin G (IgG). The kinetics and the mass transfer properties of the affinity columns were studied to determine the optimum buffer condition, flow rate, and concentration of IgG for maximum IgG adsorption. The binding capacities of all the three affinity columns were higher with zwitterionic buffer morpholinopropanesulfonic acid than with charged buffers such as tris-HCl and phosphate buffers, and the optimum pH was 6.5. The interaction of IgG with histidine immobilized CDI and epoxy disks was found to be predominantly driven by ionic interaction, while the interaction with EDA-histidine disk could be partially governed by multiple non-covalent forces of interaction. The maximum binding capacity (Qm ) of l-histidine immobilized on EDA-, CDI-, and epoxy-activated convective interaction media disks were 19.83 ± 0.25, 15.85 ± 0.18 and 12.11 ± 0.17 mg/ml of support, respectively, and the dissociation constant (Kd ) were calculated to be in the micromolar range for all the three histidyl monolithic columns. Purification of IgG from untreated human serum was also attempted, and the results indicate the high potential of this method for purification of total IgG from complex biological sources and also for separation of IgG1 from other subclasses.
authors: Centre for Bioseparation Technology, VIT University, Vellore, 632014, Tamil Nadu, India.
authors: Rajasekar R
authors: RR
authors: Prasanna
authors: Agamudi S
authors: AS
authors: Kamalanathan
authors: Mookambeswaran A
authors: MA
authors: Vijayalakshmi
mesh_terms: D000327:Adsorption; D002846:Chromatography, Affinity / Q000295:instrumentation* / Q000379:methods; D005029:Ethylenediamines / Q000737:chemistry; D006639:Histidine / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007074:Immunoglobulin G / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D013499:Surface Properties
chemical_list: D005029:Ethylenediamines; D007074:Immunoglobulin G; D006639:Histidine; C031234:ethylenediamine
keywords: CIM® disk; IgG purification; IgG subclass; l-histidine; pseudobioaffinity