Department of Pharmaceutical Sciences, University at Buffalo, The State University of New York, Amherst, New York 14260, USA.
J Pharm Sci. 2010 Apr;99(4):1697-706. doi: 10.1002/jps.21938.
Aggregation is a critical issue that hampers the development of monoclonal antibody therapeutics (Mabs). Traditionally, aggregation is considered a process in which native forms of proteins are transformed into an unstable highly associated form through an intermediate formation step. Here we describe the unfolding of an antiCD40 antibody using a folding model based on Lumry-Eyring nucleated polymerization (LENP) model. This model captures several experimental features of the thermal unfolding of this protein as studied by common in situ biophysical techniques such as circular dichroism, fluorescence spectroscopy, and turbidity measurements. According to this model, the unfolding and aggregation of the antiCD40 antibody is determined by several distinct steps that include conformational change(s) to generate aggregation prone states, reversible oligomer formation, nucleation and growth as well as their kinetics, and the formation of higher order assemblies/aggregates. Furthermore, the loss of monomer is controlled by both thermodynamic (equilibrium unfolding) and kinetic determinants of the unfolding process. This approach captures both of these rate-limiting steps. It can be concluded that this approach is sensitive to formulation conditions such as protein concentration, changes in buffer conditions, and temperature stress. The potential use of this approach in formulation development and stability testing of Mabs is discussed.
聚集是阻碍单克隆抗体治疗药物（Mabs）发展的一个关键问题。传统上，聚集被认为是蛋白质的天然形式通过中间形成步骤转化为不稳定的高度相关形式的过程。在这里，我们使用基于 Lumry-Eyring 成核聚合（LENP）模型的折叠模型描述了抗 CD40 抗体的展开。该模型捕获了通过常见的原位生物物理技术（如圆二色性、荧光光谱和浊度测量）研究该蛋白质热展开的几个实验特征。根据该模型，抗 CD40 抗体的展开和聚集是由几个不同的步骤决定的，这些步骤包括构象变化以产生易于聚集的状态、可逆的寡聚体形成、成核和生长以及它们的动力学，以及更高阶组装/聚集的形成。此外，单体的损失受展开过程的热力学（平衡展开）和动力学决定因素的控制。这种方法可以同时捕捉这两个限速步骤。可以得出结论，这种方法对制剂条件（如蛋白质浓度、缓冲条件的变化和温度应激）敏感。讨论了该方法在 Mab 的制剂开发和稳定性测试中的潜在用途。
