Boehringer Ingelheim Pharma GmbH & Co. KG, Innovation Unit, PDB, Biberach an der Riss, Germany.
Boehringer Ingelheim Pharma GmbH & Co. KG, Innovation Unit, PDB, Biberach an der Riss, Germany.
Biophys J. 2020 Mar 10;118(5):1067-1075. doi: 10.1016/j.bpj.2019.12.037. Epub 2020 Jan 16.
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 ΔH ∼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 and a Gibbs free energy of unfolding of g ∼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 ΔH and lowers the midpoint temperature T. 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.
采用差示扫描量热法（DSC）测量了重组单克隆抗体 IgG1（mAb）的热变性。DSC 图谱显示在 72°C 时存在预转变，变性焓为ΔH∼200-300 kcal/mol，在 85°C 时存在主转变，焓为∼900-1000 kcal/mol。与小的单域蛋白不同，mAb 的变性是一个复杂的反应，用多态 Zimm-Bragg 理论进行分析。对于所研究的 mAb，变性的特征是协同参数σ∼6×10 和每个氨基酸的变性自由能 g∼100 卡路里/摩尔。变性焓提供了参与变性反应的氨基酸残基数ν。平均而言，预转变涉及∼220±50 个氨基酸残基，主转变涉及∼850±30 个氨基酸残基，占所有氨基酸残基的∼90%。在存在盐酸胍的情况下进一步研究了热变性。化学变性剂降低了变性焓ΔH，并降低了中点温度 T。这两个参数都与变性剂的浓度呈线性关系。预测在 25°C 下使 mAb 变性所需的盐酸胍浓度对于预转变为 2-3 M，对于主转变为 5-7 M，这取决于 pH 值。盐酸胍与 mAb 结合具有放热结合焓，部分补偿了 mAb 变性的吸热焓。从 DSC 图谱中推断出变性时结合的盐酸胍分子数。预转变的盐酸胍-变性氨基酸比为 0.79，主转变的盐酸胍-变性氨基酸比为 0.55。预转变结合更多的变性剂分子，比主转变对变性更敏感。本研究表明，Zimm-Bragg 理论对于定量描述大的治疗性蛋白质（如单克隆抗体）的变性事件具有很强的适用性。
