Karlsruhe Institute of Technology, Institute for Functional Interfaces, Germany.
J Chromatogr A. 2012 Oct 19;1260:88-96. doi: 10.1016/j.chroma.2012.08.052. Epub 2012 Aug 23.
The effect of temperature in the range from 10°C to 40°C and comparatively low ammonium sulfate (AS) concentrations of up to 0.5M 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.
研究了温度范围为 10°C 至 40°C 以及相对较低的硫酸铵 (AS) 浓度（最高可达 0.5M）对牛血清白蛋白 (BSA) 在四种不同市售基于琼脂糖的固定相上吸附的影响。通过 Langmuir 方程拟合所确定的等温线，并通过 van't Hoff 分析计算热力学值。BSA 在色谱树脂丁基琼脂糖 4FF 上的吸附受温度影响最大；然而，通过动态柱实验对该系统进行表征时，发生了蛋白质展开效应，未折叠的 BSA 部分强烈附着在吸附剂上。不同操作条件下确定了展开部分的百分比，发现其随助溶剂盐浓度的增加而增加，但随温度的升高增加得更强。进行了温度诱导的循环吸附和解吸实验，通过纯温度控制的再生来研究丁基琼脂糖 4FF 的长期性能。在五个循环的过程中，工作容量保持稳定，但由于不完全再生，BSA 也开始在柱上积累。最后，通过完全分离溶菌酶和 BSA 证明了通过单一温度变化分离不同蛋白质的可能性。所呈现的结果表明，温度诱导的结合和洗脱可能为将 HIC 树脂的操作条件转移到降低盐浓度提供一种可能性，从而节省化学品并促进下游加工阶段的盐去除。
