Tobias M Prass, Patrick Garidel, Lars V Schäfer, Michaela Blech
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Model systems composed of two marketed therapeutic IgG1 monoclonal antibodies with identical Fc domain sequences, trastuzumab and omalizumab, were investigated with commonly used excipients arginine, glutamate, and equimolar arginine/glutamate mixtures. Protein-excipient interactions were studied using all-atom molecular dynamics (MD) simulations, which show accumulation of the excipients at specific antibody regions. Preferential excipient-interaction sites were particularly found for charged and aromatic residues and in the complementary-determining regions, with more pronounced arginine contacts for omalizumab than trastuzumab. These computational findings are in line with the more pronounced stabilizing effects of arginine observed in the long-term storage stability study. 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引用次数: 0
摘要
蛋白质制剂的开发有赖于选择能抑制蛋白质与蛋白质之间相互作用、防止聚集的辅料。经验策略包括利用强制降解或温度诱导应力(大多在加速条件下)进行理化表征,筛选多种辅料和缓冲剂组合。这些方法无法轻易提供有关辅料作用的分子间和分子内相互作用的信息。在此,我们介绍一种实验与计算相结合的方法,用于研究蛋白质与辅料之间的相互作用对制剂稳定性的影响,这种方法可以确定优先相互作用位点,从而有助于选择辅料进行实验筛选。研究了由两种具有相同 Fc 结构域序列的上市治疗用 IgG1 单克隆抗体(曲妥珠单抗和奥马珠单抗)组成的模型系统与常用辅料精氨酸、谷氨酸和等摩尔精氨酸/谷氨酸混合物的相互作用。使用全原子分子动力学(MD)模拟研究了蛋白质与辅料之间的相互作用,结果显示辅料在特定抗体区域聚集。在带电残基和芳香残基以及互补决定区发现了优先的辅料相互作用位点,奥马珠单抗的精氨酸接触比曲妥珠单抗更明显。这些计算发现与长期储存稳定性研究中观察到的精氨酸更明显的稳定作用相一致。此外,常用硅学工具预测的聚集和溶解倾向与 MD 模拟确定的优先赋形剂相互作用位点并不一致,这表明有不同的物理化学机制在起作用。
Residue-resolved insights into the stabilization of therapeutic proteins by excipients: A case study of two monoclonal antibodies with arginine and glutamate.
Protein formulation development relies on the selection of excipients that inhibit protein-protein interactions preventing aggregation. Empirical strategies involve screening many excipient and buffer combinations by physicochemical characterization using forced degradation or temperature-induced stress, mostly under accelerated conditions. Such methods do not readily provide information on the inter- and intramolecular interactions responsible for the effects of excipients. Here, we describe a combined experimental and computational approach for investigating the effect of protein-excipient interactions on formulation stability, which allows the identification of preferential interaction sites and thus can aid in the selection of excipients to be experimentally screened. Model systems composed of two marketed therapeutic IgG1 monoclonal antibodies with identical Fc domain sequences, trastuzumab and omalizumab, were investigated with commonly used excipients arginine, glutamate, and equimolar arginine/glutamate mixtures. Protein-excipient interactions were studied using all-atom molecular dynamics (MD) simulations, which show accumulation of the excipients at specific antibody regions. Preferential excipient-interaction sites were particularly found for charged and aromatic residues and in the complementary-determining regions, with more pronounced arginine contacts for omalizumab than trastuzumab. These computational findings are in line with the more pronounced stabilizing effects of arginine observed in the long-term storage stability study. Furthermore, the aggregation and solubility propensity predicted by commonly used in silico tools do not align with the preferential excipient-interaction sites identified by the MD simulations, suggesting that different physicochemical mechanisms are at play.
期刊介绍:
mAbs is a multi-disciplinary journal dedicated to the art and science of antibody research and development. The journal has a strong scientific and medical focus, but also strives to serve a broader readership. The articles are thus of interest to scientists, clinical researchers, and physicians, as well as the wider mAb community, including our readers involved in technology transfer, legal issues, investment, strategic planning and the regulation of therapeutics.