Stepwise enhancement of selectivity in hydrophobic charge-induction chromatography through grafted and dual-ligand systems

Monoclonal and polyclonal antibodies play a key role in medicine as well as in analytical biotechnology. The methods for purification of antibodies are developed fast. Hydrophobic charge induction chromatography (HCIC) is a recently developed method for protein separation based on the use of dual-mode ligands. They are designed in such a way so as to combine a molecular interaction supported by a mild hydrophobic association effect in the absence of salts. When pH is changed, the resulig and becomes ionically chargedre sulting into the desorption of the protein. HCIC shows specific advantages (such as high degree of purity and recovery) and is used in several labs and manufactures for antibody production. This paper reviewed the basic principle, development and application of HCIC. Key words: Hydrophobic charge induction chromatography; Antibody purification; Ligands

The ligands in hydrophobic charge induction chromatography (HCIC) are hydrophobic and ionisable. Thus, the pH is crucial for the separation performance in HCIC, especially for elution. However, it is difficult to obtain the microscopic information in HCIC through experimental means. In this work, molecular dynamics simulations are performed to examine the effect of pH on elution and protein conformational transition in HCIC, using a 46-bead β-barrel coarse-grained model protein and an HCIC adsorbent pore model constructed in an earlier work. Corresponding experiments are carried out for the validation of simulation results, using lysozyme and MEP Hypercel. Both the activities and fluorescence of lysozyme are examined to evaluate the conformational transition. The simulations indicate that the elution efficiency of protein increases with decreasing pH value in a non-linear manner. This is qualitatively consistent with the experimental results. MD simulations indicate that protein unfolding occurs in elution at all pH values. However, the experimental data show that the activity and conformation of lysozyme is independent of pH of the elution buffer. The microscopic information from simulation shows that protein unfolding is mainly observed on the adsorbent surface, but it cannot be detected in the experiments that only probe the proteins in the bulk liquid.

Development and application of hydrophobic charge-induction chromatography for bioseparation

The adsorption behaviors of hydrophobic charge induction chromatography (HCIC) adsorbents with different functional ligands were investigated with immunoglobulin of egg yolk (IgY) as a model antibody. The adsorption isotherm and retention behavior in the column were studied, and the influences of the ligand structure and the pH on the adsorption were discussed. The results indicated that the pI of the target protein and pKa of HCIC ligand are the important parameter to determine the maximum adsorption pH of HCIC adsorbent, and high adsorption of IgY was found at pH 5 for all five adsorbents tested. Some differences could be found for different HCIC adsorbents, and the ligand structure influenced pH effect on the binding/elution of target protein. 2-mercapto-1-methyl-imidazole (MMI) ligand with a sulfone group showed a high adsorption capacity and strong pH-sensitivity, which would be more suitable for antibody purification. Moreover, the retention experiments indicated that IgY could be efficiently eluted from the adsorbents with 4-mercapto-ethyl-pyridine (MEP) or MMI as the ligand at acid conditions, while 2-mercapto-benzimidazole (MBI) ligand showed some difficulties on the elution. The retention study would help in defining not only the effective pH of elution for a given protein but also the elution efficiency of a given adsorbent.

Displacement chromatography of proteins on hydrophobic charge induction adsorbent column

Initial purification of recombinant botulinum neurotoxin fragments for pharmaceutical production using hydrophobic charge induction chromatography

Hydrophobic charge induction chromatography (HCIC) is an adsorption chromatography combining hydrophobic interaction in adsorption with electrostatic repulsion in elution. The method has been successfully applied in the separation and purification of antibodies and other proteins. However, little is understood about protein conformational transition and the dynamic process within adsorbent pores. In the present study, a pore model is established to represent the realistic porous adsorbent composed of matrix and immobilized HCIC ligands. Protein adsorption, desorption, and conformational transition in the HCIC pore and its implications to the separation performance are shown by a molecular dynamics simulation of a 46-bead beta-barrel coarse-grained model protein in the adsorbent pore. Repeated adjustment of both protein position and orientation is observed before reaching a stable adsorption. Once the protein is adsorbed, there is a dynamic equilibrium between unfolding and refolding. The effect of hydrophobic interaction strength between protein and ligands on adsorption phenomena is then examined. Strong hydrophobic interaction, representing the presence of high-concentration lyotropic salt in mobile phase, can speed up the adsorption but cause protein unfolding more significantly. On the contrary, weak hydrophobic interaction, representing the absence of a lyotropic salt or the presence of a chaotropic agent, can reserve native protein conformation but does not lead to stable adsorption. In the elution, protein unfolding occurs due to simultaneous hydrophobic adsorption and electrostatic repulsion in the opposite directions. When the protein has been desorbed, the conformational transition between unfolded and native protein is still observed due to the long-range nature of electrostatic interaction. The simulation has provided molecular insight into protein conformational transition in the whole HCIC process, and it would be beneficial to the rational design of ligands and parameter optimizations for high-performance HCIC.

A new purification process for goose immunoglobulin IgY(ΔFc) with hydrophobic charge-induction chromatography

Application of cyclodextrin-based eluents in hydrophobic charge-induction chromatography: Elution of antibody at neutral pH

Antibody separation by hydrophobic charge induction chromatography

Hydrophobic charge-induction chromatography (HCIC) is a novel technology for antibody separation. In this paper, the immunoglobulin of egg yolk (IgY) was chosen as a model antibody to investigate the effects of salt on HCIC. The adsorption behavior of antibody IgY on several HCIC adsorbents as a function of salt concentration was studied using adsorption isotherms and adsorption kinetics. The hydrodynamic diameters and ζ potentials of IgY at various salt concentrations were also determined. It was found that the saturated adsorption capacities increased linearly with increasing salt concentration because of the improvement of hydrophobic interactions between IgY and the HCIC ligands. The pore diffusion model was used to evaluate the dynamic adsorption process. The total effective diffusivity (De′) showed a maximum value at an ammonium sulfate concentration of 0.2 M. The results indicate salt-promoted adsorption under the appropriate concentration due to a reduction of protein size and the enhancement of hydrophobic interactions between IgY and the HCIC ligand. Therefore, the addition of a proper amount of salt is beneficial for antibody adsorption in the HCIC process.

Molecular dynamics simulation of the effect of ligand homogeneity on protein behavior in hydrophobic charge induction chromatography

Immunoglobulin of egg yolk (IgY) was separated from fresh hen eggs by three steps, water dilution, salt-out precipitation, and hydrophobic charge induction chromatography (HCIC). In the purification step, HCIC was used as a novel technology and two chromatographic operation modes, packed bed and expanded bed, were compared to improve the separation efficiency. For packed bed mode, IgY were separated with the purity of 92% and the recovery of 58.4% at the operation condition of pH 7.5 and sample (total protein concentration of 11.6 mg/mL, IgY purity of 31%) loading of 0.2 mL/mL adsorbent. For expanded bed mode, a similar purity of 90% and the recovery of 59.5% were found at the fluid velocity of 500 cm/h and same sample loading of 0.24 mL/ mL adsorbent at pH 7.5. The results demonstrated that HCIC in expanded bed mode is a potential platform technology for antibody separation both in high efficiency and low cost.

5-Aminobenzimidazole as new hydrophobic charge-induction ligand for expanded bed adsorption of bovine IgG

Multifunctionalization of RC membrane via combining surface initiated RAFT polymerization with thiolactone chemistry for enhanced antibody recovery