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

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.

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

In recent years, bottom-up approach has become the popular method of choice for large scale analysis of complex proteome samples. Peptide fractionation determines the efficiency of the bottom-up method and often the resolving power of reverse phase liquid chromatography (RPLC) is insufficient for efficient protein identification in case of complex biological samples. To overcome the inherent limitation of proteomics associated with sample complexity, we evaluated fast flow metal chelate methacrylate monolithic system - CIM (Convective Interaction Media) disk chelated with Cu(II) for targeted affinity selection of histidine-containing peptides. Initially the Cu(II)-IMAC using CIM disk was evaluated using tryptic digest of protein mixtures of 8 model proteins and was found to be highly efficient in capturing His-containing peptides with high degree of specificity and selectivity. Further the efficiency of His-peptide enrichment using CIM-IMAC was also demonstrated using complex biological samples like total Escherichia coli cell lysate. The analysis of the Cu(II)-IMAC retained peptides from tryptic digests of model protein mixture and E. coli not only demonstrated a significant reduction in sample complexity but also subsequently enabled the identification of additional peptides. His-peptide enrichment also enabled the identification of low abundant proteins that were not detected in the analysis of total E. coli digest.

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

Dynamic binding capacity (DBC) of commercial metal-chelate methacrylate monolith-convective interaction media (CIM) was performed with commercial human immunoglobulin G (IgG) (Cohn fraction II, III). Monoliths are an attractive stationary phase for purification of large biomolecules because they exhibit very low back pressure even at high flow rates and flow-unaffected binding properties. Adsorption of IgG onto CIM-IDA disk immobilized with Cu(2+), Ni(2+) and Zn(2+) were studied with Tris-acetate (TA), phosphate-acetate (PA) and MMA (MES, MOPS and acetate) buffer systems at different flow rates. Adsorption and elution of IgG varied with different buffers and adsorption of IgG was maximum with MMA buffer. Adsorption of human IgG from Cohn fractions (II, III) was high when Cu(2+) was used as ligand. CIM-IDA disk showed dynamic binding capacity in the range of 14-16 mg/ml with Cu(2+) and 7-9 mg/ml with Ni(2+) for human IgG with MMA buffer. In the case of CIM-IDA-Zn(2+) column, the binding capacity was only about 0.5mg/ml of support. Different desorption strategies like lowering of pH and increasing of competitive agent were also studied to achieve maximum recovery. Chromatographic runs with human serum and mouse ascites fluid were also carried out with metal chelate methacrylate monolithic disk and the results indicate the potential of this technique for polyclonal human IgG and monoclonal IgG purification from complex biological samples.