Pseudo-mercaptoethyl pyridine functionalized polyhedral oligomeric silsesquioxane-graphene composite via thiol-ene click reaction for highly selective purification of antibody.

In this work, we prepared a novel graphene-based composite by combination of mercapto-end capped polyhedral oligomeric silsesquioxane (POSS-SH) with graphene oxide (GO) through covalent bonding process. The composite was further conjugated with 4-vinyl pyridine (4-VP) through thiol-ene click reaction to fabricate pseudo-mercaptoethyl pyridine functionalized POSS-graphene composite (G-POSS-S-VP). The physicochemical properties were confirmed via FT-IR, TGA, XPS, and TEM characterizations and a high ligand density of ca. 87.3 µmol m-2 was obtained. G-POSS-S-VP composite displayed high selectivity toward antibody, i.e., immunoglobulin G (IgG) in this case based on the hydrophobic charge induced chromatography (HCIC) mechanism. A high adsorption efficiency of ca. 90% and large adsorption capacity of 500 mg g-1 were achieved for IgG at pH 7.4. At the same time, the non-specific adsorptions of co-existing proteins on graphene substrate were obviously hindered. The practical applicability of G-POSS-S-VP composite as adsorbent was verified by selective separation of IgG from human serum. SDS-PAGE assay and MALDI-TOF-MS analysis clearly demonstrated the successful isolation of IgG with high purity.

Evaluation of hydrophobic charge-induction ligand efficiency for protein adsorption in one single cycle.

Ligand is an essential part of the cost of adsorbent preparation, which needs to be carefully selected and evaluated. In this paper, we introduced ligand efficiency (Le) with three levels (recovery, preparation and cost) to form a selection strategy for evaluation of the efficiency of hydrophobic charge-induction ligand. These functions were calculated from static/dynamic binding capacity, desorption efficiency, coupling efficiency and ligand cost. Nine kinds of ligand were used to demonstrate this strategy. The coupling efficiency was determined by preparing the adsorbents with different kinds and densities of ligand. These adsorbents were characterized by FT-IR, SEM. Then adsorption equilibrium, adsorption kinetics, and frontal adsorption experiments were used to test the adsorption and desorption performance of these adsorbents. Finally, Les of recovery, preparation and cost were calculated. The results showed there were apparent differences in Les between ligand types and densities under static and dynamic adsorption conditions. 4FF-Tryptophan with 52 µmol/g adsorbent had the best performance with the lowest static/dynamic Le of recovery, preparation and ligand cost. Compared with those methods evaluated by static saturated adsorption capacity or dynamic binding capacity at 10% breakthrough, the selection strategy based on ligand efficiency is more suitable for subsequent research and industrial amplification.

A novel multimodal chromatography based single step purification process for efficient manufacturing of an E. coli based biotherapeutic protein product.

Methionine oxidized, reduced and fMet forms of a native recombinant protein product are often the critical product variants which are associated with proteins expressed as bacterial inclusion bodies in E. coli. Such product variants differ from native protein in their structural and functional aspects, and may lead to loss of biological activity and immunogenic response in patients. This investigation focuses on evaluation of multimodal chromatography for selective removal of these product variants using recombinant human granulocyte colony stimulating factor (GCSF) as the model protein. Unique selectivity in separation of closely related product variants was obtained using combined pH and salt based elution gradients in hydrophobic charge induction chromatography. Simultaneous removal of process related impurities was also achieved in flow-through leading to single step purification process for the GCSF. Results indicate that the product recovery of up to 90.0% can be obtained with purity levels of greater than 99.0%. Binding the target protein at pH

Evaluation of mixed-mode chromatographic resins for separating IgG from serum albumin containing feedstock.

Mixed-mode chromatography has been focused as a cost-effective new technique for antibody purification. In this study, four mixed-mode resins with N-benzyl-N-methyl ethanol amine, 2-benzamido-4-mercaptobutanoic acide, 4-mercapto-ethyl-pyridine and phenylpropylamine as the ligands were tested and the multi-functional interactions between ligand and protein were discussed. Immunoglobulin G (IgG), bovine serum albumin (BSA) and the binary mixture of BSA and IgG were used as the model feedstock to compare the separation behaviors by pH gradient elution. The comparison analysis showed mixed-mode resin with N-benzyl-N-methyl ethanol amine as the ligand had the best ability to separate IgG and BSA. The results indicated that for four resins tested ionic interaction might play the dominant role in the separation of IgG and BSA while the hydrophobic interactions and hydrogen bonding have some subsidiary effects. The pH stepwise elution and sample loading were optimized to improve the IgG purification from serum albumin containing feedstock. High purity (92.3%) and high recovery (95.6%) of IgG were obtained. The results indicated that mixed-mode chromatography would be a potential option for antibody purification with the control of loading and elution conditions.

Study on fast purification of monoclonal antibody against rhTF_(243) protein with the technique of HCIC / 中国免疫学杂志

Objective:To develop a two-step method for purification of monoclonal antibody rhTF243 protein from mouse ascites by using hydrophobic charge induction chromatography(HCIC) and affinity chromatography with protein A sepharose CL-4B.Methods:The ascites was first purified by HCIC after centrifugation and filtration.Then the fraction containing the protein of interest was directly purified by affinity chromatography with protein A sepharose CL-4B.Results:The purity of the obtained monoclonal antibody was up to 97% with recovery of 73% and of high activity.Conclusion:The method for purification of monoclonal antibody is developed using HCIC and Protein A affinity chromatography and the obtained antibodies are of high purity and activity.