Technology trends in antibody purification.

After 35 years of development, immobilized metal ion affinity chromatography (IMAC) has evolved into a popular protein purification technique. This review starts with a discussion of its mechanism and advantages. It continues with its applications which include the purification of histidine-tagged proteins, natural metal-binding proteins, and antibodies. IMAC used in conjunction with mass spectroscopy for phosphoprotein fractionation and proteomics is also covered. Finally, this review addresses the developments, limitations, and considerations of IMAC in the biopharmaceutical industry.

Immobilized metal ion affinity chromatography: a review on its applications

Amino acids and peptides are often used as “model” segments of proteins for studying their behavior in various types of environments, and/or elaborating functional surfaces. Indeed, though the protein behavior is much more complex than that of their isolated segments, knowledge of the binding mode as well as of the chemical structure of peptides on metal or oxide surfaces is a significant step toward the control of materials in a biological environment. Such knowledge has considerably increased in the past few years, thanks to the combination of advanced characterization techniques and of modeling methods. Investigations of biomolecule–surface interactions in water/solvent environments are quite numerous, but only in a few cases is it possible to reach an understanding of the molecule–(water)–surface interaction with a level of detail comparable to that of the UHV studies. This contribution aims at reviewing the recent data describing the amino acid and peptide interaction with metal or oxide surfaces in ...

Adsorption of Amino Acids and Peptides on Metal and Oxide Surfaces in Water Environment: A Synthetic and Prospective Review.

Affinity chromatography: A versatile technique for antibody purification.

Alternative Affinity Ligands for Immunoglobulins

Characterization of metal chelate methacrylate monolithic disk for purification of polyclonal and monoclonal immunoglobulin G.

Affinity selection of histidine-containing peptides using metal chelate methacrylate monolithic disk for targeted LC-MS/MS approach in high-throughput proteomics.

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 65 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 1983 ± 025, 1585 ± 018 and 1211 ± 017 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

Development of L-histidine immobilized CIM(®) monolithic disks for purification of immunoglobulin G.

Immobilized metal-ion affinity (IMA) adsorption is a collective term that is used to include all kinds of adsorptions where the metal ion serves as the characteristic and most es- sential part of adsorption center. Of all the IMA techniques, immobilized metal-affinity chromato graphy (IMAC) has been gaining popularity as the choice of purification technique for proteins. IMAC represents a separation technique that is primarily useful for proteins with natural surface exposed-histidine residues and for recombinant proteins with engineered his- tidine tag. This review also gives insight into other nonchromatographic applications of IMA adsorption such as immobilized metal-ion affinity gel electrophoresis (IMAGE), immobi- lized metal-ion affinity capillary electrophoresis (IMACE), and immobilized metal-ion affin- ity partitioning (IMAP).

Immobilized metal-ion affinity systems for recovery and structure–function studies of proteins at molecular, supramolecular, and cellular levels

Purified monoclonal antibodies (mAb) have been used in therapeutics and some analytical procedures. Purification of mAb by use of high-throughput anion-exchange methacrylate monolithic systems has been attempted in this work. Monolithic macroporous convective interaction media (CIM) with diethylaminoethyl (DEAE) and ethylene diamine (EDA) as anion-exchange ligands were used and evaluated for purification of anti-glycophorin-A IgG1 mouse mAbs from cell culture supernatant (CCS) after precipitation with 50% ammonium sulfate. The adsorption and elution of mAb from the CCS on CIM-DEAE and CIM-EDA disks were studied with three different buffer systems, acetate, MOPS (3-(N-morpholino)propanesulfonic acid), and Tris, to study the effect of the nature of buffer ions and to find the optimum buffer conditions for purification of mAb. The optimum buffers for purification of mAb using CIM-DEAE and CIM-EDA were 50 mM acetate buffer, pH 5.1 and 20 mM Tris buffer, pH 8.0, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and enzyme-linked immunosorbent assay (ELISA) showed the antibody fractions obtained were highly pure, with high antigen-binding efficiency. High specific activity with purification factors of 130 ± 34 (unretained fraction with acetate buffer) and 74 ± 13 (fraction eluted with Tris buffer containing 0.6 M NaCl) was obtained for IgG1 using the CIM-DEAE and CIM-EDA disks, respectively. The results indicate that rapid separation and efficient recovery of high-purity anti-glycophorin-A mAbs could be achieved by use of anion-exchange CIM disks.

Rajasekar R. Prasanna

Papers

Characterization of metal chelate methacrylate monolithic disk for purification of polyclonal and monoclonal immunoglobulin G.

Affinity selection of histidine-containing peptides using metal chelate methacrylate monolithic disk for targeted LC-MS/MS approach in high-throughput proteomics.

Development of L-histidine immobilized CIM(®) monolithic disks for purification of immunoglobulin G.

Immobilized metal-ion affinity systems for recovery and structure–function studies of proteins at molecular, supramolecular, and cellular levels

Purification of Monoclonal Antibodies from Cell-Culture Supernatant by Use of Anion-Exchange Convective Interaction Media (CIM) Monolithic Columns