Department of Bio-Science, Nagahama Institute of Bio-Science and Technology, 1266 Tamura, Nagahama 526-0829, Japan.
Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba 305-8566, Japan.
Int J Mol Sci. 2023 Jul 27;24(15):12042. doi: 10.3390/ijms241512042.
Antibody aggregation, followed by acid denaturation and neutralization of pH, is one of the reasons why the production of therapeutic monoclonal antibodies (mAbs) is expensive. Determining the structural details of acid-denatured antibodies is important for understanding their aggregation mechanism and for antibody engineering. Recent research has shown that monoclonal antibodies of human/humanized immunoglobulin G1 (IgG1) become smaller globules at pH 2 compared to their native structure at pH 7. This acid-denatured species is unstable at pH 7 and prone to aggregation by neutralization of pH. Small-angle X-ray scattering (SAXS) data have revealed an acid-induced reduction in the subpeaks in Kratky plot, indicating conformational changes that can lead to aggregation. The subpeaks are well resolved at pH > 3 but less pronounced at pH ≤ 2. One of the weakened subpeaks indicates loosely organized inter-region (Fab-Fab and Fab-Fc) correlations due to acid denaturation. However, the structural origin of the other subpeak (called peak in this study) has not been established because its region could represent the various inter-region, inter-domain, and intra-domain correlations in IgG1. In this study, we aimed to untangle the effects of domain-domain correlations on Kratky's peak based on the computed SAXS of the crystal structure of IgG1. The peak appeared in the static structure and was more prominent in the Fc region than in the Fab or isolated domains. Further brute-force analysis indicated that longer domain-domain correlations, including the inter-region, also positively contribute to Kratky's peak. Thus, the distortion of the Fc region and a longer inter-region correlation initiate acid denaturation and aggregation.
抗体聚集，随后是酸变性和 pH 值中和，是治疗性单克隆抗体 (mAb) 生产成本高的原因之一。确定酸变性抗体的结构细节对于了解其聚集机制和抗体工程至关重要。最近的研究表明，与人源/人源化免疫球蛋白 G1 (IgG1) 的单克隆抗体在 pH 2 时比在 pH 7 时形成更小的球蛋白。这种酸变性的物种在 pH 7 时不稳定，并且在 pH 值中和时容易聚集。小角 X 射线散射 (SAXS) 数据显示，在 Kratky 图中，酸诱导的亚峰减少，表明可能导致聚集的构象变化。在 pH > 3 时，亚峰得到很好的分辨，但在 pH ≤ 2 时则不太明显。一个减弱的亚峰表明由于酸变性导致区域间（Fab-Fab 和 Fab-Fc）相关性松散。然而，另一个亚峰（在本研究中称为 峰）的结构起源尚未确定，因为其 区域可能代表 IgG1 中的各种区域间、域间和域内相关性。在这项研究中，我们旨在根据 IgG1 晶体结构的计算 SAXS 来解开结构域-结构域相关性对 Kratky's 峰的影响。该峰出现在静态结构中，在 Fc 区域比在 Fab 或分离的结构域中更为突出。进一步的暴力分析表明，较长的结构域-结构域相关性，包括区域间相关性，也对 Kratky 的 峰有积极贡献。因此，Fc 区域的扭曲和较长的区域间相关性引发了酸变性和聚集。
