Higher-Order Structure and Solution Interactions of Antibodies

单克隆抗体（mAb）是一类快速增长的蛋白质治疗剂。 为了使治疗性施用所需的体积最小化，mAb通常以100 mg/ml的浓度配制，这远高于现有颗粒大小测定方法的范围。 虽然沉降速度分析超离心已成为测量稀溶液中的高阶结构的金标准，但我们实验室最近开发的非理想沉降速度（SV）新技术为表征mAb溶液更接近制剂条件提供了新的机会。原则上，这允许人们在比以前可能的高10倍的蛋白质浓度下进行研究。 除了抗体与其抗原靶标的明显的高亲和力相互作用之外，还有三种其他类型的抗体相互作用在药物制剂中是令人感兴趣的。 首先，短距离和长距离的静电力和流体动力学调节蛋白质的距离分布在解决方案中。这可能导致净排斥或吸引相互作用，而不形成蛋白质复合物。虽然非常微妙，但这种相互作用是宏观溶液行为和长期蛋白质稳定性的关键。它们通常通过沉降、扩散和热力学维里系数的非理想系数来测量。 第二种类型的抗体相互作用是它们通过形成短寿命的可逆蛋白质-蛋白质复合物而自我缔合的倾向。 这些都与高溶液粘度有关。非理想性和自缔合性质都是浓度依赖性的，并且在稀溶液中不能很好地测量。 我们与阿斯利康合作，使用一组单克隆抗体，确定IgG mAb的非理想SV浓度限值为45 mg/mL。 我们能够证明，这种高度浓缩的样品，它是可能可靠地测量非理想系数的沉降。 同时，新的非理想SV方法提供了一种简单而灵敏的手段来表征抗体的自缔合。 在理论工作中，我们分析了弱自关联对不同生物物理方法测量非理想系数的影响。 这澄清了不相容的热力学参考系的习惯使用，例如，在静态和动态光散射，并解释了严重不一致的非理想系数经常在文献中发现。 为了克服这个问题，我们已经扩展了我们的全球多方法分析（GMMA）软件，允许全球分析非理想从不同的数据源。 这弥合了不同技术之间灵敏度和分辨率的差距，并允许使用自洽的参考系，将非理想参数与统计流体动力学理论联系起来。 除了抗体的表征，弱相互作用和非理想性的GMMA为DMAS在浓缩蛋白质溶液领域的相关兴趣提供了实验平台，包括眼透镜晶状体蛋白。 最后，对于抗体制剂非常重要的第三类蛋白质相互作用是来自错误折叠蛋白质的长寿命聚集体，其可以随时间形成。 这些药物具有潜在的免疫原性，因此必须对痕量药物进行监测并向FDA报告。 为了检查非理想SV的较高浓度限度对痕量蛋白质二聚体定量限的影响，我们之前与John Schiel博士（NIST）的实验室合作开展了一项热变性NISTmAb参比抗体的研究。 我们已经进行了混合实验的天然和热变性的NISTmAb在平行的非理想SV和尺寸排阻色谱。 我们已经开发了一个合适的数据分析协议的痕量分析使用非理想SV。 基于这些结果，我们设计了控制实验来加强技术的比较。

Monoclonal antibodies (mAbs) are a rapidly growing class of protein therapeutics. To minimize the volume required for therapeutic administration, mAbs are typically formulated at concentrations on the order of 100 mg/ml, which is far above the range of existing particle sizing methods. While sedimentation velocity analytical ultracentrifugation has become the gold standard for measuring higher-order structures in dilute solutions, new opportunities have arisen for characterizing mAb solutions much closer to formulation conditions with the new technique of nonideal sedimentation velocity (SV), recently developed in our laboratory. In principle this allows one to study at 10fold higher protein concentrations than previously possible. Beyond the obvious high-affinity interaction of antibodies with their antigen target, there are three additional types of antibody interactions that are of interest in pharmaceutical formulations. First, short- and long-range electrostatic and hydrodynamic forces modulate protein distance distributions in solution. This may result in net repulsive or attractive interactions without formation of protein complexes. Though very subtle, such interactions are key to macroscopic solution behavior and long-term protein stability. They are commonly measured through nonideality coefficients of sedimentation, diffusion, and thermodynamic virial coefficients. A second type of antibody interactions is their propensity to self-associate through formation of short-lived, reversible protein-protein complexes. These have been associated with high solution viscosity. Both nonideality and self-association properties are concentration-dependent and cannot be measured well in dilute solution. Using a panel of monoclonal antibodies, in collaboration with AstraZeneca, we have established concentration limits of nonideal SV for IgG mAbs to be 45 mg/mL. We were able to show that with such highly concentrated samples it is possible to reliably measure nonideality coefficients of sedimentation. Simultaneously the new nonideal SV approach provides a simple and sensitive means to characterize self-association of antibodies. In theoretical work we have analyzed the impact of weak self-association on the measurement of nonideality coefficients with different biophysical methods. This has clarified the customary use of incompatible thermodynamic reference frames, for example, in static and dynamic light scattering, and explains grossly inconsistent nonideality coefficients frequently found in the literature. To overcome this problem, we have extended our global multi-method analysis (GMMA) software to allow global analysis of nonideality from different data sources. This bridges gaps in sensitivity and resolution between different techniques, and permits the use of a self-consistent reference frame that links nonideality parameters up with statistical fluid dynamics theory. Beyond the characterization of antibodies, GMMA of weak interactions and nonideality provides an experimental platform for related interests in DMAS in the field of concentrated protein solutions, including eye lens crystallins. Finally, a third category of protein interactions that are very important for antibody formulations are long-lived aggregates from misfolded protein that can form with time. These are potentially immunogenic and therefore trace quantities must be monitored and reported to FDA. In order to examine what impact the higher concentration limits of nonideal SV have on the limits of quantitation for trace protein dimers, we have previously embarked on a study of heat-denatured NISTmAb reference antibody, in collaboration with the laboratory of Dr. John Schiel (NIST). We have carried out mixing experiments of native and heat-denatured NISTmAb in parallel by nonideal SV and size exclusion chromatography. We have developed a suitable data analysis protocol for trace analysis using nonideal SV. Based on the results, we have designed control experiments to enhance comparison of techniques.