Responsive membranes for hydrophobic interaction chromatography: The effect of salt ions
Ranil Wickramasinghe, Zizhao Liu, Xianghong Qian
The use of environmentally responsive membranes for hydrophobic interaction chromatography (HIC) enables high recovery and high resolution during protein purification. Further the possibility of protein fractionation also exists. Here poly N-vinylcaprolactam (PVCL) has been grafted from the surface of commercially available regenerated cellulose membranes (0.45 μm pore size) via atom transfer radical polymerization. PVCL displays a lower critical solution temperature (LCST) at around 32 °C in DI water. The observed LCST of PVCL is a strong function of solution ionic strength and type of salt ion present. Increasing ionic strength leads to a decrease in the LCST. Above the LCST the grafted polymer chains collapse, dehydrate and appear more hydrophobic promoting protein adsorption. Below the LCST the grafted chains swell, hydrate and promote protein desorption.
Optimization of this novel system requires a systematic investigation of different salt types and concentrations. Here dynamic binding capacities of our PVCL modified membranes were determined at different pH, salt type and concentration at room temperature with two model proteins, a human immunoglobulin4, IgG4, and bovine serum albumin (BSA). In addition, BSA adsorption isotherms in the presence of monovalent (Na+, NH4+), divalent (Mg2+, Zn2+) and trivalent (Al3+) sulfate solutions were compared in order to investigate the effect of the cation). Selected BSA binding capacity data (left hand side y-axis) and recovery data (right hand side y-axis) are given in the figure below. As can be seen the ionic strength and type of salt not only affect the binding capacity, but also the recovery during elution. Finally, the responsiveness of the PVCL ligand synthesized in solution as well as grafted on silicon wafers was investigated. Molecular dynamics simulations were also conducted to elucidate the salt effect on the PVCL ligand.