Laboratory of Engineering Thermodynamics, University of Kaiserslautern, Kaiserslautern, Germany.
J Chromatogr A. 2011 Jul 22;1218(29):4720-6. doi: 10.1016/j.chroma.2011.05.063. Epub 2011 May 27.
Adsorption of native as well as mono-, di-, and tri-PEGylated lysozyme on Toyopearl Gigacap S-650M in sodium phosphate buffer is studied by isothermal titration calorimetry and by independent adsorption equilibrium measurements at pH 6 and 25°C. The production and separation of PEGylated lysozyme is discussed. Two different PEG sizes are used (5 kDa and 10 kDa) which leads to six different forms of PEGylated lysozyme which were systematically studied. The sodium chloride concentration is varied according to the elution conditions in the production process. The specific enthalpy of adsorption Δh(p)(ads) is determined from the calorimetric and the adsorption equilibrium data. It was found to be exothermal and constant with increasing adsorber loading for native lysozyme. For all PEGylated forms there is an influence of the adsorber loading on Δh(p)(ads) which is found to become more important with increasing PEGylation degree (total molecular weight of conjugated PEG). At low loadings the adsorption of all PEGylated lysozyme forms is exothermal. With increasing loading the adsorption becomes less exothermal and for the species with higher PEGylation degree also endothermal effects are observed at higher loadings. A thermodynamic analysis is carried out by which the enthalpic and entropic contributions to the binding constants are quantified. The findings are discussed on a molecular level. The results provide insight into the adsorption mechanisms of polymer-modified proteins on chromatographic resins.
采用等温热滴定法和独立吸附平衡测量法，在 pH 6 和 25°C 条件下，研究了天然、单、二和三聚乙二醇化溶菌酶在 Toyopearl Gigacap S-650M 上的吸附。讨论了 PEG 化溶菌酶的生产和分离。使用了两种不同的 PEG 分子量（5 kDa 和 10 kDa），得到了六种不同形式的 PEG 化溶菌酶，对其进行了系统研究。根据生产过程中的洗脱条件，改变了氯化钠浓度。从量热法和吸附平衡数据确定了吸附特异性焓变Δh(p)(ads)。对于天然溶菌酶，它是放热的，并且随着吸附剂负载的增加而保持不变。对于所有 PEG 化形式，Δh(p)(ads)受吸附剂负载的影响，随着 PEG 化程度（共轭 PEG 的总分子量）的增加，这种影响变得更加重要。在低负载下，所有 PEG 化溶菌酶形式的吸附都是放热的。随着负载的增加，吸附变得不那么放热，对于具有较高 PEG 化程度的物质，在较高负载下也观察到吸热效应。通过热力学分析，量化了结合常数的焓和熵贡献。讨论了分子水平上的结果。这些结果为聚合物修饰蛋白质在色谱树脂上的吸附机制提供了深入的了解。
