Abstract：
The fibrillation of therapeutic peptides can present significant quality concerns and poses challenges for manufacturing and storage. A fundamental understanding of the mechanisms of fibrillation is critical for the rational design of fibrillation-resistant peptide drugs and can accelerate product development by guiding the selection of solution-stable candidates and formulations. The studies reported here investigated the effects of structural modifications on the fibrillation of a 29-residue peptide (PepA) and two sequence modified variants (PepB, PepC). The C-terminus of PepA was amidated, whereas both PepB and PepC retained the carboxylate, and Ser16 in PepA and PepB was substituted with a helix-stabilizing residue, α-aminoisobutyric acid (Aib), in PepC. In thermal denaturation studies by far-UV CD spectroscopy and fibrillation kinetic studies by fluorescence and turbidity measurements, PepA and PepB showed heat-induced conformational changes and were found to form fibrils, whereas PepC did not fibrillate and showed only minor changes in the CD signal. Pulsed hydrogen-deuterium exchange mass spectrometry (HDX-MS) showed a high degree of protection from HD exchange in mature PepA fibrils and its proteolytic fragments, indicating that most of the sequence had been incorporated into the fibril structure and occurred nearly simultaneously throughout the sequence. The effects of the net peptide charge and formulation pH on fibrillation kinetics were investigated. In real-time stability studies of two formulations of PepA at pH\'s 7.4 and 8.0, analytical methods detected significant changes in the stability of the formulations at different time points during the study, which were not observed during accelerated studies. Additionally, PepA samples were withdrawn from real-time stability and subjected to additional stress (40 °C, continuous shaking) to induce fibrillation; an approach that successfully amplified oligomers or prefibrillar species previously undetected in a thioflavin T assay. Taken together, these studies present an approach to differentiate and characterize fibrillation risk in structurally related peptides under accelerated and real-time conditions, providing a model for rapid, iterative structural design to optimize the stability of therapeutic peptides.
摘要：
治疗性肽的原纤化可能存在显著的质量问题,并对制造和储存提出挑战。对纤维化机制的基本理解对于抗纤维化肽药物的合理设计至关重要，并且可以通过指导选择溶液稳定的候选物和制剂来加速产品开发。此处报道的研究调查了结构修饰对29残基肽（PepA）和两个序列修饰变体（PepB，PepC）。PepA的C末端被酰胺化，而PepB和PepC都保留了羧酸盐，PepA和PepB中的Ser16被螺旋稳定残基取代，α-氨基异丁酸（Aib），在PepC。在通过远UVCD光谱进行的热变性研究和通过荧光和浊度测量进行的原纤化动力学研究中，PepA和PepB显示出热诱导的构象变化，并被发现形成原纤维，而PepC没有纤维化，仅显示CD信号的微小变化。脉冲氢-氘交换质谱（HDX-MS）在成熟的PepA原纤维及其蛋白水解片段中显示出高度的HD交换保护作用，表明大多数序列已被掺入原纤维结构中，并且在整个序列中几乎同时发生。研究了净肽电荷和制剂pH对原纤维化动力学的影响。在pH=7.4和8.0的两种PepA制剂的实时稳定性研究中，分析方法在研究期间的不同时间点检测到制剂稳定性的显著变化，在加速研究期间没有观察到。此外,PepA样品从实时稳定性中取出，并经受额外的应力(40℃，连续摇动）以诱导纤颤；一种成功扩增先前在硫黄素T测定中未检测到的寡聚体或原纤丝物质的方法。一起来看,这些研究提出了一种在加速和实时条件下区分和表征结构相关肽纤颤风险的方法，提供一个快速的模型，迭代结构设计以优化治疗性肽的稳定性。
