Suitability of commercial hydrophobic interaction sorbents for temperature-controlled protein liquid chromatography under low salt conditions
Müller, T. / Franzreb, M. (2012)
Journal of Chromatography A 1260 (2012), 88–96
- Date: 2012
Müller, T. / Franzreb, M. (2012): „Suitability of commercial hydrophobic interaction sorbents for temperature-controlled protein liquid chromatography under low salt conditions“. In: Journal of Chromatography A 1260 (2012), 88–96
The effect of temperature in the range from 10 °C to 40 °C and comparatively low ammonium sulfate (AS) concentrations of up to 0.5 M on the adsorption of bovine serum albumin (BSA) on four different commercially available sepharose-based stationary phases was investigated. The determined isotherms were fitted by the Langmuir equation, and thermodynamic values were calculated by van’t Hoff analysis.
The adsorption of BSA onto the chromatographic resin Butyl Sepharose 4FF showed the strongest temperature influence; however, protein unfolding effects occurred when characterizing this system by dynamic column experiments, with an unfolded BSA fraction strongly attached to the sorbent.
The percentage of the unfolding fraction was determined for different operating conditions and found to increase with the concentration of the cosmotropic salt, but even stronger with increasing temperature. Temperature-induced cyclic adsorption and desorption experiments were carried out to investigate the long-term performance of Butyl Sepharose 4FF by applying purely temperature-controlled regeneration.
Over a period of five cycles, the working capacity remained stable, but BSA also started to accumulate on the column due to incomplete regeneration. Finally, the possibility to fractionate different proteins with a single temperature shift was shown by the complete separation of lysozyme and BSA. The results presented indicate that temperature-induced binding and elution may offer a possibility to shift the operation conditions of HIC resins toward reduced salt concentrations, thus saving chemicals and facilitating salt removal in further downstream processing stages.