Chromatography has been widely used for the separation of biomolecules during the past decades. Generally, the separation essence of adsorptive chromatography is based on the binding modes between the target protein and the functional ligands on the adsorbents. Varying adsorption and desorption modes correspond to different chromato-graphic processes, for instance, the electrostatic adsorption and high-salt-concentration desorption for ion exchange chromatography (1–3), hydrophobic binding and low-salt-concentration desorption for hydrophobic interaction chromatography (4,5), the biospecific combination and analogous compound replacement for affinity chromatography, etc (6,7). Among varying factors, the functional ligands play the most important role in the binding of target protein and determine the separation efficiency for chromatographic process.
Mixed-mode chromatography (MMC) is an exciting new technology for bioseparation, which can provide multimodal interactions between the ligand and target protein (8). The mixed-mode ligands typically contain a combination of multiple binding modes like ion exchange, hydrophobic interactions and hydrogen bonding, which results in a variety of protein-ligand interactions and often leads to high selectivity for efficient separation. Furthermore, the binding of the desired protein is often achieved at the moderate conductivity of the feedstock, which is suitable for primary capture without prior dilution or addition of lyotropic salts (9). Streamline Direct HST, Capto MMC, and Capto adhere are typical commercial mixed-mode adsorbent from GE Healthcare. Hamilton et al. (10) and Gao et al. (11–14) used benzylamine as the functional ligand to prepare an anion-exchange mixed-mode adsorbent for expanded bed adsorption. For the MMC, the multimodal protein-ligand interactions are critical, which determines the adsorption and separation efficiency of target protein.
In 1998, Burton and Harding (15) developed a new kind of MMC, named hydrophobic charge induction chromatography (HCIC), providing the hydrophobic binding and charge-induced desorption. The ligands of HCIC might be specially designed, normally combining hydrophobic, thiophilic, and electrostatic interactions. The most well known HCIC adsorbent is MEP HyperCel which was developed by Pall Corporation. The 4-mercapto-ethyl-pyridine (MEP) ligand contains a pyridine ring with the pKa of 4.8 and a sulfur atom in the spacer arm. The protein can be adsorbed at neutral pH by the thiophilic and hydrophobic interactions with sulfur atom and pyridine ring on the ligand, and desorbed under the electrostatic repulsion between the protein and the charged ligands when the pH is less than the pKa of MEP and pI of the target protein (16). It was found that HCIC ligands could bind selectively to the Fc domains of immunoglobulin and showed a potential application for antibody purification as the
cost-effective alternative to Protein A affinity chromatography (17–21). Due to the multimodal interactions between the HCIC ligand and the target protein, the adsorption and separation behaviors are quite complicated and the design of the ligand is often empirical. Several researchers have studied the interaction between HCIC ligands and target protein (22–24). However, the mechanismic understandings are still limited and more studies on the effects of ligand structure are necessary which would certainly improve the rational design of novel HCIC ligands.
In the previous works (25–27), with three mercaptohe-terocycles (4-mercapto-ethyl-pyridine (MEP), 2-mercapto-1-methyl-imidazole (MMI), and 2-mercapto-benzimidazole (MBI)) as functional ligands, four HCIC adsorbents (Cell-TuC-AB-MEP, Cell-TuC-DVS-MEP, Cell-TuC-DVS-MMI, and Cell-TuC-DVS-MBI) were prepared with Cell-TuC as cellulose composite matrix and two kinds of activation methods, allylbromide activation or divinylsulfone activation. In the present work, with immunoglobulin of egg yolk (IgY) as the model antibody, the adsorption behaviors of these HCIC adsorbents were investigated as the function of pH. The adsorption isotherms and retention behavior in the column were studied systematically, and the influences of the ligand structure and pH were discussed.
The effects of ligand structure and pH on the adsorption behaviors of IgY onto five HCIC adsorbents were investigated with the adsorption isotherm and retention experiments. It was found that the adsorption behavior of IgY on the HCIC adsorbents are strongly depended on the ligand structure and pH. The ligand structure influences pH effect on the binding=elution of target protein. The pI of the target protein and pKa of HCIC ligand are the important parameter to determine the maximum adsorption pH of HCIC adsorbent. The highest adsorption capacities of IgY were found at pH 5, near the pKa of ligands and around the pI of IgY. Sulfone group on the spacer arm could enhance the binding of IgY but cause some difficulties on the elution. MMI ligand showed a high adsorption capacity and strong pH-sensitivity, which would be more suitable for antibody purification. MBI ligand might be not suitable for HCIC due to hard elution either in acid condition or in alkali condition. Moreover, the results indicated that the retention study can help define not only the effective pH of elution for a given protein but also the elution efficiency of a given adsorbent. The acid condition about pH 3.4$3.6 would be suitable for the effective elution of IgY and alkali condition for CIP. In general, a better understanding of the molecular interactions between the ligand and the protein would certainly improve the design of new ligands and the applications of HCIC for antibody separation.