热力学研究辅料与蛋白药物之间的相互作用

摘要

目的：研究不同辅料（氨基酸、糖类、非离子型表面活性剂）对蛋白药物IgG1单抗热稳定性的影响，探讨辅料与蛋白之间的相互作用，判断辅料与蛋白之间是否存在结合作用。方法通过差示扫描量热法（ differential scanning calorimetry， DSC）获得蛋白的变性温度（ Tm ）或蛋白某一结构域的变性温度。采用等温滴定量热法（ isothermal titration calorimetry ，ITC）测定蛋白与不同辅料之间的相互作用。结果带负电荷的氨基酸可以明显降低IgG1单抗的变性温度（ Tm ＞9℃），其他辅料作用不明显（ Tm ＜1℃）。不同pH下辅料对蛋白稳定性的影响不同，在pH＝5时带负电荷的氨基酸对IgG1单抗热稳定性的影响大于pH＝7时。 ITC实验数据显示各种辅料和IgG1单抗的滴定等温线近乎一条直线。结论 IgG1单抗与辅料之间不存在特异性相互作用，带负电荷的氨基酸可以明显降低IgG1单抗的变性温度，应该归功于2者之间的静电相互作用。%Objective To determine the effects of different excipients ( amino acids, carbohydrates and nonionic surfactants) on thermal stability of the IgG1 monoclonal antibody, and to examine the interactions between the excipients and the protein.Methods Differential scanning calorimetry ( DSC) was used to study thermal stability of the protein in different solutions and got information on the solubility of the unfolded forms of the protein.Isothermal titration calorimetry ( ITC) was used to examine the binding interactions between the excipients and the protein.ResuIts Negatively charged amino acids could significantly reduce the denaturation temperature (Tm) of IgG1( Tm >9 ℃), and other excipients didn’t have a major effect ( Tm <1℃).Excipients shared different impacts on thermal stability of the IgG1 monoclonal antibody under different pH, and negatively charged amino acids result in a much lower Tm at pH 5 than at pH 7.The ITC binding isotherms of different excipients (including polysorbate 20 and 80) and IgG1 were almost straight lines, while there was strong binding interaction between polysorbate 20 or 80 and Human Serum Albumin (HSA).ConcIusion The results suggest that there is no binding interaction between these studied excipients and the IgG1 monoclonal antibody; instead electrostatic interactions seem to play a leading role between the excipients and the IgG1 monoclonal antibody.