Anal Bioanal Chem 406:4987–4995īorges E, Euerby M, Collins C (2012) Characterization of a mixed-mode reversed-phase/cation-exchange stationary phase prepared by thermal immobilization of poly(dimethylsiloxane) onto the surface of silica. J Chromatogr A 469:3–27įu Y, Mo H-Y, Gao W, Hong J-Y, Lu J, Li P, Chen J (2014) Affinity selection-based two-dimensional chromatography coupled with high-performance liquid chromatography-mass spectrometry for discovering xanthine oxidase inhibitors from Radix Salviae Miltiorrhizae. Melander WR, El Rassi Z, Horváth C (1989) Interplay of hydrophobic and electrostatic interactions in biopolymer chromatography: effect of salts on the retention of proteins. Zhao G, Dong X-Y, Sun Y (2009) Ligands for mixed-mode protein chromatography: principles, characteristics and design. McLaughlin LW (1989) Mixed-mode chromatography of nucleic acids. The results indicate that the dual-function mixed-mode stationary phase prepared in this study may aid in the development of new two-dimensional liquid chromatography technology with a single column for intensive proteomic studies, and it may also find applications in recombinant protein drug production since it can save column costs and simplify the biotechnological processes.
The mass recovery, purity, and specific bioactivity obtained for the purified recombinant human interferon-γ were 87.2 %, 92.4 %, and 2.8 × 10 7 IU/mg, respectively, in IEC mode, and 83.4 %, 95.2 %, and 4.3 × 10 7 IU/mg, respectively, in HIC mode. On the basis of this dual-function mixed-mode chromatography column, a new two-dimensional liquid chromatography technology with a single column system was also developed in this study, and was used to renature and purify recombinant human interferon-γ from inclusion bodies. The results show that electrostatic interaction of the ligand with proteins must match the hydrophobicity of the ligand, which is an important factor to prepare the dual-function stationary phase. In addition, the effects on protein separation of different ligand structures in the dual-function stationary phase and the pH of the mobile phase used were also investigated in detail. The results indicate that the novel dual-function mixed-mode column in many cases can replace the use of two individual WCX and HIC columns. Protein mass and bioactivity recoveries of more than 96 % can be achieved in both HIC mode and IEC mode using this column. In comparison with the conventional WCX and HIC columns, the results were satisfactory and acceptable. The resolution and selectivity of the stationary phase were evaluated in both HIC mode and IEC mode using protein standards. It can be used to separate proteins in both ion-exchange chromatography (IEC) mode and HIC mode. The new system displays both hydrophobic interaction chromatography (HIC) character in a high salt concentration mobile phase, and weak cation exchange (WCX) chromatography character in a low salt concentration mobile phase. A novel dual-function mixed-mode stationary phase based on poly(glycidyl methacrylate- co-ethylene dimethacrylate) microspheres was synthesized by thiol–ene click chemistry and characterized by infrared spectroscopy and elemental analysis.
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