Higher-Order Structure and Solution Interactions of Antibodies

单抗是一种快速发展的蛋白质治疗药物。为了最大限度地减少给药所需的体积，单抗的浓度通常在100毫克/毫升左右，这远远超出了现有的颗粒测量方法的范围。随着沉降速度分析超速离心法已经成为测量稀溶液中高阶结构的金标准，我们实验室最近开发的非理想沉降速度(SV)新技术为更接近配方条件的单抗溶液的表征提供了新的机会。原则上，这允许人们在比以前可能的蛋白质浓度高10倍的情况下进行研究。 除了抗体与其抗原靶标的明显高亲和力相互作用外，还有三种额外类型的抗体相互作用在药物配方中是有意义的。首先，短距离和长距离的静电和流体动力调节蛋白质在溶液中的距离分布。这可能导致净排斥或吸引相互作用而不形成蛋白质复合体。虽然非常微妙，但这种相互作用是宏观溶液行为和蛋白质长期稳定性的关键。它们通常通过非理想的沉积系数、扩散系数和热力学维里系数来测量。抗体相互作用的第二种类型是它们通过形成短暂的、可逆的蛋白质-蛋白质复合体而自我结合的倾向。这些都与高溶液粘度有关。非理想性和自缔合性质都与浓度有关，在稀溶液中不能很好地测量。 使用一组单抗，与阿斯利康合作，我们确定了Ig G单抗的非理想SV的浓度限制为45 mg/m L。我们能够证明，对于如此高浓度的样品，有可能可靠地测量沉积的非理想系数。同时，新的非理想SV方法提供了一种简单而灵敏的方法来表征抗体的自结合。 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。这阐明了不相容热力学参照系的习惯用法，例如，在静态和动态光散射中，并解释了文献中经常发现的严重不一致的非理想性系数。为了克服这个问题，我们扩展了我们的全球多方法分析(GMMA)软件，允许对不同数据源的非理想性进行全局分析。这弥补了不同技术之间在灵敏度和分辨率上的差距，并允许使用一个自洽的参照系，将非理想性参数与统计流体动力学理论联系起来。除了抗体的特性外，弱相互作用和非理想性的GMMA为包括眼晶状体晶体蛋白在内的浓缩蛋白质溶液领域的相关兴趣提供了一个实验平台。 最后，第三类对抗体配方非常重要的蛋白质相互作用是错误折叠的蛋白质随时间形成的长寿命聚集体。这些物质具有潜在的免疫原性，因此必须对微量进行监测并向FDA报告。为了检验非理想SV的较高浓度极限对微量蛋白质二聚体定量极限的影响，我们先前与John Schiel(NIST)博士的实验室合作开展了热变性NISTmAb参考抗体的研究。我们用非理想SV和大小排斥层析平行进行了天然和热变性NISTmAb的混合实验。我们开发了一种适用于使用非理想SV进行踪迹分析的数据分析协议。根据结果，我们设计了对照实验，以加强技术的比较。

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.