The stability and aggregation of NIST monoclonal antibody (NISTmAb) were investigated by hydrogen/deuterium exchange mass spectrometry (HDX-MS), differential scanning calorimetry (DSC), and nano-differential scanning fluorimetry (nanoDSF). NISTmAb was prepared in eight formulations at four different pHs (pH 5, 6, 7, and 8) in the presence and absence of 150 mM NaCl and analyzed by the three methods. The HDX-MS results showed that NISTmAb is more conformationally stable at a pH near its isoelectric point (pI) in the presence of NaCl than a pH far from its pI in the absence of NaCl. The stabilization effects were global and not localized. The midpoint temperature of protein thermal unfolding transition results also showed the C_H2 domain of the protein is more conformationally stable at a pH near its pI. On the other hand, the onset of aggregation temperature results showed that NISTmAb is less prone to aggregate at a pH far from its pI, particularly in the absence of NaCl. These seemingly contradicting results, higher conformational stability yet higher aggregation propensity near the pI than far away from the pI, can be explained by intramolecular and intermolecular electrostatic repulsion using Lumry-Eyring model, which separates folding/unfolding equilibrium and aggregation event. The further a pH from the pI, the higher the net charge of the protein. The higher net charge leads to greater intramolecular and intermolecular electrostatic repulsions. The greater intramolecular electrostatic repulsion destabilizes the protein and the greater intermolecular electrostatic repulsion prevents aggregation of the protein molecules at pH far from the pI.

中文翻译：

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HDX-MS、DSC 和 nanoDSF 揭示分子内和分子间静电排斥对 NISTmAb 稳定性和聚集的影响

通过氢/氘交换质谱 (HDX-MS)、差示扫描量热法 (DSC) 和纳米差示扫描荧光法 (nanoDSF) 研究 NIST 单克隆抗体 (NISTmAb) 的稳定性和聚集性。在存在和不存在 150 mM NaCl 的情况下，在四种不同pH 值（pH  5、6、7 和 8）的八种制剂中制备 NISTmAb ，并通过三种方法进行分析。HDX-MS 结果表明，在 NaCl 存在下，NISTmAb在其等电点 (p I )附近的 ap H处比远离其 p I的 ap H处的构象更稳定在没有氯化钠的情况下。稳定效应是全球性的，而不是局部的。蛋白质热解折叠转变结果的中点温度也表明蛋白质的CH ₂结构域在接近其p I的pH 下构象更稳定。另一方面，聚集温度的开始结果表明 NISTmAb 在远离其 p I的 ap H处不太容易聚集，特别是在没有 NaCl 的情况下。这些看似矛盾的结果，更高的构象稳定性和更高的聚集倾向，靠近 p I比远离 p I，可以通过使用 Lumry-Eyring 模型的分子内和分子间静电排斥来解释，该模型将折叠/展开平衡和聚集事件分开。离 p I越远的p H，蛋白质的净电荷越高。较高的净电荷导致较大的分子内和分子间静电排斥。较大的分子内静电排斥使蛋白质不稳定，较大的分子间静电排斥防止蛋白质分子在远离 p I的 p H处聚集。