Molecular dynamics simulation of the effect of ligand homogeneity on protein behavior in hydrophobic charge induction chromatography.
ABSTRACT Hydrophobic charge induction chromatography (HCIC) is an adsorption chromatography combining hydrophobic interaction in adsorption with electrostatic repulsion in elution. Ligand density has significant effects on protein adsorption behavior, but little is understood about the effect of ligand homogeneity on surface morphology of ligands, protein conformational transition and dynamics within adsorbent pore due to the lack of microscopic experimental techniques. In the present study, a coarse-grained adsorbent pore model established in an earlier work is used to represent the actual porous adsorbent composed of matrix and immobilized HCIC ligands. Two adsorbent pores with different ligand distributions are constructed by adjusting the coupling sites, denoted as L1 and L2. In L1 the ligands are bonded uniformly while in L2 the ligands are arranged in lines in the axial direction and thus exhibit a heterogeneous distribution. Protein adsorption, desorption, and conformational transition in both L1 and L2 are shown by molecular dynamics simulations of a 46-bead beta-barrel coarse-grained model protein within the adsorbent pore models. The simulations indicate that ligand homogeneity has significant effect on both the irreversibility and the dynamics of adsorption while no obvious effect on protein conformation distribution. In comparison with L1, L2 leads to irreversible and slow adsorption, indicating the strict requirement of a suitable protein orientation to reach stable adsorption. The simulations have provided new insight into the microscopic behavior of HCIC, which would be beneficial to the rational design of adsorbents and parameter optimization for high-performance HCIC.
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ABSTRACT: Aggregate level is a key factor when assessing quality attributes of antibody product, since the aggregation might impact the biological activity of biopharmaceutical. Effective separation methods are usually essential in manufacturing processes. In this study, three mixed-mode resins, i.e. Capto adhere and two home-made resins with benzylamine and butylamine as the functional ligands (named BA and AB), were used to investigate the separation performances of antibody aggregate removal processes. In addition, two traditional resins (Q Sepharose FF and Phenyl Sepharose 6 FF) were also investigated to explore the role of molecular interaction in the aggregate removal. The results indicated that the removal efficiency was highly dependent on the mass loading. With the sample load of 50mg/ml resin both Capto adhere and BA resins can significantly reduce the aggregate level from 20.5% to 2.6% and 2.4%, respectively, while a relatively low degree of aggregate reduction was found with the traditional resins. The research indicates that both hydrophobic interaction and electrostatic interaction are critical for the aggregate removal and the cooperativity of different molecular interactions is important for the effective aggregates removal with mixed-mode resins.Journal of Chromatography A 04/2013; · 4.61 Impact Factor