Biophysical properties of antibody-based biopharmaceuticals are a critical part of their release criteria. In this context, finding the appropriate formulation is equally important as optimizing their intrinsic biophysical properties through protein engineering, and both are mutually dependent. Most previous studies have empirically tested the impact of additives on measures of colloidal stability, while mechanistic aspects have usually been limited to only the thermodynamic stability of the protein. Here we emphasize the kinetic impact of additives on the irreversible denaturation steps of immunoglobulins G (IgG) and their antigen-binding fragments (Fabs), as these are the key committed steps preceding aggregation, and thus especially informative in elucidating the molecular parameters of activity loss. We examined the effects of ten additives on the conformational kinetic stability by differential scanning calorimetry (DSC), using a recently developed three-step model containing both reversible and irreversible steps. The data highlight and help to rationalize different effects of the additives on the properties of full-length IgG, analyzed by onset and aggregation temperatures as well as by kinetic parameters derived from our model. Our results further help to explain the observation that stabilizing mutations in the antigen-binding fragment (Fab) significantly affect the kinetic parameters of its thermal denaturation, but not the aggregation properties of the full-length IgGs. We show that the proper analysis of DSC scans for full-length IgGs and their corresponding Fabs not only helps in ranking their stability in different formats and formulations, but provides important mechanistic insights for improving the conformational kinetic stability of IgGs.

基于抗体的生物药物的生物物理特性是其释放标准的关键部分。在这种情况下，寻找合适的配方与通过蛋白质工程优化其内在的生物物理特性同等重要，两者相互依赖。以前的大多数研究已通过经验测试了添加剂对胶体稳定性测量的影响，而机械方面通常仅限于蛋白质的热力学稳定性。这里我们强调动力学添加剂对免疫球蛋白G（IgG）及其抗原结合片段（Fabs）不可逆变性步骤的影响，因为这些是聚集之前的关键步骤，因此在阐明活性丧失的分子参数方面尤其有用。我们使用最近开发的包含可逆和不可逆步骤的三步模型，通过差示扫描量热法（DSC）检查了十种添加剂对构象动力学稳定性的影响。数据突出并帮助合理化了添加剂对全长IgG特性的不同影响，并通过开始和聚集温度以及从我们的模型得出的动力学参数进行了分析。我们的结果进一步有助于解释这一观察结果，即抗原结合片段（Fab）中的稳定突变会显着影响其热变性的动力学参数，但不会影响全长IgG的聚集特性。我们表明，对全长IgG及其相应Fab进行DSC扫描的正确分析不仅有助于对它们在不同形式和制剂中的稳定性进行排名，而且还为改善IgG的构象动力学稳定性提供了重要的机理见解。