Higher-Order Structure and Solution Interactions of Antibodies

抗体的高阶结构和溶液相互作用

基本信息

批准号：
10263002
负责人：
PETER SCHUCK
金额：
$ 7.98万
依托单位：
NATIONAL INSTITUTE OF BIOMEDICAL IMAGING AND BIOENGINEERING
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10263002
关键词：
Affinity Antibodies Antigen Targeting Behavior Biotechnology Categories Collaborations Computer software Crystalline Lens Crystallins Data Analyses Data Sources Diffusion Electrostatics Formulation Gold Higher Order Chromatin Structure Immunoglobulin G Laboratories Link Liquid substance Literature Measurement Measures Methods Molecular Conformation Molecular Sieve Chromatography Monitor Monoclonal Antibodies Particle Size Pharmaceutical Preparations Pharmacologic Substance Physiological Property Proteins Protocols documentation Reporting Resolution Safety Sampling Serum Techniques Therapeutic Therapeutic antibodies Thermodynamics Time Viscosity Work analytical ultracentrifugation base biophysical techniques design dimer experimental study hydrodynamic flow immunogenic interest light scattering misfolded protein protein complex sedimentation coefficient sedimentation velocity standard measure theories therapeutic protein

项目摘要

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.

单克隆抗体（mAb）是一种快速生长的蛋白质疗法。为了最大程度地减少治疗施用所需的体积，MAB通常以100 mg/ml的浓度为浓度，远高于现有粒度方法的范围。虽然沉积速度分析性超速离心已成为测量稀释溶液中高阶结构的金标准，但已经出现了新的机会，它以新的非理想沉积速度（SV）的新技术来表征更接近配方条件的MAB溶液（SV）。从原则上讲，这允许人们以比以前可能更高的蛋白质浓度研究。除了抗体与其抗原靶标的明显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引起了人们的关注。首先，短期和远程静电和流体动力调节溶液中的蛋白质距离分布。这可能会导致净排斥或有吸引力的相互作用，而不会形成蛋白质复合物。尽管非常微妙，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。它们通常是通过沉积，扩散和热力学病毒系数的非理想系数来测量的。第二种抗体相互作用是它们通过形成短寿命，可逆蛋白质蛋白复合物形成自我关联的倾向。这些与高溶液粘度有关。非理想性和自我关联特性都是浓度依赖性的，在稀释溶液中不能很好地测量。使用一组单克隆抗体，与阿斯利康合作，我们确定了非理想的SV的浓度限制为IgG mAbs为45 mg/ml。我们能够证明，使用如此高度浓缩的样品，可以可靠地测量沉积的非理想系数。同时，新的非理想SV方法为表征抗体的自我关联提供了一种简单而敏感的手段。在理论工作中，我们分析了弱自我关联对使用不同生物物理方法测量非理想系数的影响。这阐明了习惯使用不兼容的热力学参考帧，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系数。为了克服这个问题，我们扩展了全球多方法分析（GMMA）软件，以允许来自不同数据源的非理想性分析。这弥合了不同技术之间的敏感性和分辨率的差距，并允许使用将非理想参数与统计流体动力学理论联系起来的自洽参考框架。除了抗体的表征外，弱相互作用和非理想性的GMMA为浓缩蛋白质溶液（包括眼镜结晶蛋白）的DMA相关兴趣提供了一个实验平台。最后，对于抗体制剂非常重要的第三类蛋白质相互作用是可能随时间形成的错误折叠蛋白的长寿命。这些是潜在的免疫原性，因此必须监测痕量并将其报告给FDA。为了检查非理想SV的较高浓度限制对痕量蛋白二聚体的定量限制的影响，我们以前曾与John Schiel博士（NIST）合作进行了热增生的Nistmab参考抗体的研究。我们通过非理想的SV和尺寸排除色谱法并行进行了天然和热贬值的Nistmab的混合实验。我们已经开发了一种合适的数据分析协议，用于使用非理想的SV进行痕量分析。根据结果，我们设计了控制实验以增强技术的比较。