The thermal unfolding of a recombinant monoclonal antibody IgG1 (mAb) was measured with differential scanning calorimetry (DSC). The DSC thermograms reveal a pretransition at 72°C with an unfolding enthalpy of ΔHcal ∼200–300 kcal/mol and a main transition at 85°C with an enthalpy of ∼900–1000 kcal/mol. In contrast to small single-domain proteins, mAb unfolding is a complex reaction that is analyzed with the multistate Zimm-Bragg theory. For the investigated mAb, unfolding is characterized by a cooperativity parameter σ ∼6 × 10−5 and a Gibbs free energy of unfolding of gnu ∼100 cal/mol per amino acid. The enthalpy of unfolding provides the number of amino acid residues ν participating in the unfolding reaction. On average, ν∼220 ± 50 amino acids are involved in the pretransition and ν∼850 ± 30 in the main transition, accounting for ∼90% of all amino acids. Thermal unfolding was further studied in the presence of guanidineHCl. The chemical denaturant reduces the unfolding enthalpy ΔHcal and lowers the midpoint temperature Tm. Both parameters depend linearly on the concentration of denaturant. The guanidineHCl concentrations needed to unfold mAb at 25°C are predicted to be 2–3 M for the pretransition and 5–7 M for the main transition, varying with pH. GuanidineHCl binds to mAb with an exothermic binding enthalpy, which partially compensates the endothermic mAb unfolding enthalpy. The number of guanidineHCl molecules bound upon unfolding is deduced from the DSC thermograms. The bound guanidineHCl-to-unfolded amino acid ratio is 0.79 for the pretransition and 0.55 for the main transition. The pretransition binds more denaturant molecules and is more sensitive to unfolding than the main transition. The current study shows the strength of the Zimm-Bragg theory for the quantitative description of unfolding events of large, therapeutic proteins, such as a monoclonal antibody.

中文翻译：

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单克隆 IgG1 抗体的热和化学展开：多态 Zimm-Bragg 理论的应用

使用差示扫描量热法 (DSC) 测量重组单克隆抗体 IgG1 (mAb) 的热解折叠。DSC 热谱图显示在 72°C 发生预转变，展开焓为 ΔHcal ～200-300 kcal/mol，在 85°C 下发生主转变，焓为 900-1000 kcal/mol。与小的单域蛋白质相比，mAb 的解折叠是一个复杂的反应，用多态 Zimm-Bragg 理论进行分析。对于所研究的 mAb，展开的特征在于协同参数 σ∼6 × 10−5 和 gnu 展开的吉布斯自由能 ∼100 cal/mol 每个氨基酸。去折叠焓提供了参与去折叠反应的氨基酸残基 ν 的数量。平均而言，ν∼220 ± 50 个氨基酸参与预转换，ν∼850 ± 30 个氨基酸参与主转换，约占所有氨基酸的 90%。在盐酸胍存在下进一步研究了热解折叠。化学变性剂降低解折叠焓 ΔHcal 并降低中点温度 Tm。这两个参数都与变性剂的浓度呈线性关系。在 25°C 下展开 mAb 所需的盐酸胍浓度预计为 2–3 M（预转变）和 5–7 M（主要转变），随 pH 值变化。盐酸胍以放热结合焓与 mAb 结合，这部分补偿了吸热 mAb 的解折叠焓。解折叠时结合的盐酸胍分子的数量由 DSC 热谱图推导出来。结合的盐酸胍与未折叠氨基酸的比率对于预转换为 0.79，对于主转换为 0.55。预转变结合更多的变性分子并且比主要转变对去折叠更敏感。目前的研究显示了 Zimm-Bragg 理论在定量描述大型治疗性蛋白质（如单克隆抗体）展开事件方面的优势。