Insights into protein-polysorbate interactions analysed by means of isothermal titration and differential scanning calorimetry
Insights into protein-polysorbate interactions analysed by means of isothermal titration and differential scanning calorimetry
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DOI：
10.1007/s00249-009-0404-6
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发表时间：
2009-06-01
影响因子：
2
通讯作者：
Garidel, Patrick
中科院分区：
文献类型：
--
作者：
Hoffmann, Claudia;Blume, Alfred;Garidel, Patrick
配制成高蛋白浓度液体溶液的治疗性蛋白质对化学和物理降解非常敏感。特别是避免蛋白质聚集体的形成对产品质量非常重要。为了稳定蛋白质治疗剂的胶体性质，使用各种赋形剂。特别是洗涤剂聚山梨酯20和80是常见的。然而，去污剂保护蛋白质免于聚集的机制尚不清楚。本研究探讨了聚山梨酯20和80与不同蛋白质的相互作用：溶菌酶，牛血清白蛋白（BSA）和免疫球蛋白。通过等温滴定量热法（ITC）研究去污剂与蛋白质的相互作用和结合。从ITC导出结合时的热力学参数（Δ H：焓变化，Δ S：熵和Δ G：自由能）以及结合常数K（α）。通过差示扫描量热法（DSC）评估蛋白质在去污剂存在下的热稳定性。结果表明，两种去污剂均与BSA结合，K（a）在8和12 x 10（3）M-1之间，Δ H为-50至-60 kJ/mol（25 A ℃）。1 - 2个去污剂分子与BSA结合。这两种洗涤剂的存在下诱导BSA的热变性性质的弱稳定。然而，聚山梨酯20和80与溶菌酶和免疫球蛋白的相互作用是相当可忽略的。浓度高达2 mM的去污剂的存在对热容曲线没有影响，未观察到天然构象的不稳定或稳定。
Therapeutic proteins formulated as liquid solutions at high protein concentration are very sensitive to chemical and physical degradation. Especially avoiding the formation of protein aggregates is very crucial for product quality. In order to stabilize the colloidal properties of protein therapeutics various excipient are used. Especially the detergents polysorbate 20 and 80 are common. However, the mechanism upon which the detergents protect the protein from aggregation is not really known. The present study investigates the interaction of polysorbate 20 and 80 with different proteins: lysozyme, bovine serum albumin (BSA) and an immunoglobulin. The interaction and binding of the detergents to the proteins is investigated by isothermal titration calorimetry (ITC). From ITC the thermodynamic parameters (Delta H: change in enthalpy, Delta S: entropy and Delta G: free energy) upon binding are derived as well as the binding constant K (a). The thermal stability of the proteins in the presence of the detergent is assessed by differential scanning calorimetry (DSC). The results show that both detergents bind to BSA with K (a) between 8 and 12 x 10(3) M-1 with Delta H -50 to -60 kJ/mol (25A degrees C). One to two detergent molecules bind to BSA. The presence of both detergents induces a weak stabilisation of the thermal denaturation properties of BSA. However, the interaction of polysorbate 20 and 80 with lysozyme and the immunoglobulin is quite negligible. The presence of the detergents up to a concentration of 2 mM has no impact on the heat capacity curve neither a destabilisation nor a stabilisation of the native conformation is observed.