Preparative Chromatography of Biological Macromolecules

描述(由申请人提供)：拟议的研究旨在提高从大蛋白质到病毒的大生物大分子和组件的制备性色谱分离的效率。在获得许可的生物治疗药物中，越来越多地发现了更大的蛋白质，如单抗，而病毒和病毒样颗粒则通过层析纯化作为疫苗和基因治疗载体。实现这种改进的方法是建立在对大溶质在色谱介质中的吸附、解吸和传输机制的更好的定量和定性理解的基础上的。在这个寻找结构-性能关系的框架内将解决的具体目标将使用色层测量和固定相表面的修饰，以及几种成像方法，即。电子断层扫描确定孔网络的三维结构，扫描共聚焦显微镜监测溶质进入固定相的吸收，原子力显微镜观察吸附层的结构。从这些方法获得的见解将与适当的建模方法相结合，包括模拟溶质在孔网络中的扩散，模拟吸收到色谱介质中，以及胶体模拟吸附相互作用。实验和建模方法将协同使用，并使用几种蛋白质和病毒在各种商业固定相上进行评估，这些固定相通常用于制备层析实践。拟议研究的成功完成将有助于为特定应用选择固定相的有效和合理的方法，并有助于设计新的固定相的方法。

DESCRIPTION (provided by applicant): The proposed research seeks to improve the efficacy of preparative chromatographic separations of large biomacromolecules and assemblies, ranging from large proteins to viruses. Larger proteins such as monoclonal antibodies are increasingly found among licensed biotherapeutics, while viruses and virus-like particles are purified chromatographically as vaccines and as gene therapy vectors. The approaches that will be used to accomplish such improvements are based on developing a better quantitative and qualitative understanding of the mechanisms of adsorption, desorption and transport of large solutes in chromatographic media. The specific aims that will be addressed within this framework of seeking structure-performance relationships will employ chromatographic measurements and modification of stationary-phase surfaces, as well as several imaging methods, viz. electron tomography to determine the 3D structure of pore networks, scanning confocal microscopy to monitor uptake of solutes into stationary phases, and atomic force microscopy to observe the structure of adsorbate layers. The insights obtained from these methods will be coupled with appropriate modeling approaches, including simulations of solute diffusion in pore networks, modeling of uptake into chromatographic media, and colloidal modeling of adsorbate interactions. The experimental and modeling approaches will be used synergistically, and evaluated using several proteins and viruses on a variety of commercial stationary phases that are routinely used in preparative chromatography practice. Successful completion of the proposed research will facilitate efficient and rational approaches to stationary-phase selection for a particular application, and aid in methods for designing novel stationary phases.