High Selectivity and Capacity Bioseparations using Hybrid Displacement-Multimodal Systems

提出的更新项目建立在我们在离子交换（IEX）和疏水相互作用（HIC）系统中置换色谱的重要前期工作以及我们对多模态（MM）系统中蛋白质相互作用的新理解的基础上，以创建一种混合分离技术，该技术将产生高选择性和高容量的蛋白质分离。这项工作将基于我们实验室的最新进展，这些进展表明，选择性置换剂的使用可以显著提高羟基磷灰石（HA）和其他多模态（MM）色谱系统的选择性。我们将采用高通量筛选（HTS）和分子模拟的工具，为这种选择行为的本质提供基本的见解，并解决与MM系统的选择性驱替剂设计相关的几个关键问题。将采用机器人高通量驱替剂筛选技术筛选MM系统中的选择性驱替剂，并评估蛋白质、驱替剂结构、流动相特性和驱替剂浓度的影响。这项工作将利用我们小组先前开发的蛋白质和置换物文库来建立模型蛋白质对和潜在的置换物，以供进一步分析。还将生成多组分吸附等温线，以研究这些驱替剂-蛋白质- mm系统中的竞争结合、协同相互作用和滞后效应。将进行列研究，以确定这种混合方法可以实现的增强选择性和性能的水平。还将进行动态亲和图分析，以检验是否可以从质量作用的角度来理解增强的选择性和位移-解吸转变。广泛的实验和理论技术将被用来检查选择性置换与固定的MM配体的相互作用。表面等离子体共振光谱将研究动力学效应，而等温滴定量热法将提供深入了解MM系统中选择性位移过程中涉及的潜在机制。将进行一系列分子模拟来研究选择性置换剂与MM表面的相互作用，并为各种分子间相互作用的重要性提供基本见解。多组分吸附的分子模拟也将用小蛋白质在置换剂和盐反离子溶液中进行，以进一步了解MM系统中竞争结合和置换过程的机制。最后，将使用工业合作伙伴提供的复杂生物混合物来评估在MM系统中解决具有挑战性的生物制药分离的选择性取代剂的使用。目前迫切需要开发新技术，以满足安全、低成本的下一代生物疗法日益增长的挑战，这些生物疗法包括蛋白质（如抗体、酶）、多糖（如肝素、透明质酸）和基于细胞的技术。拟议的项目将产生一种强大的新型混合分离技术来满足这一需求。在拟议的研究中获得的见解将有助于开发和实施改进的选择性置换MM色谱系统，用于纯化治疗性蛋白质。这将潜在地消除对昂贵的亲和色谱系统的需求，并将减少所需的下游处理步骤的数量，对生产生物药品的成本产生重大影响。本项目中描述的基础研究也将为复杂多组分混合物的疏/亲水性和结合改性提供重要见解，这将为深入了解这些影响重要的一系列系统（例如生物分离，生物传感器，生物材料）建立一个新的平台。显然，如果提案获得成功，将对生物加工和若干健康相关领域产生重大而广泛的影响。本研究也将对化学工程专业本科研究生及高中学生的教育产生重要影响。克莱默实验室在培养具有色谱生物处理一流培训的化学工程师方面有着悠久的历史。这个培训将继续对参与这个项目的学生进行。拟议的研究将在生物技术和跨学科研究中心进行，为学生提供优秀的多学科培训机会，并接触广泛的实验和模拟技术。这个项目的研究也将被纳入PI最近开发的化学工程高级实验室色谱实验以及色谱分离过程课程中。最后，在这个项目中进行的模拟，以及分子相互作用的概念部分，将激发分子馆项目的新方面，它使用动画电影来教授和激励各级学生关于分子迷人世界的知识。

1134341CramerThe proposed renewal project builds upon our significant body of prior work in displacement chromatography in ion exchange (IEX) and hydrophobic interaction (HIC) systems as well as our new understanding of protein interactions in multimodal (MM) systems to create a hybrid separations technology that will produce highly selective and high capacity protein separations. This work will is based upon recent advances in our lab that have demonstrated that the use of selective displacers can result in dramatically enhanced selectivity in hydroxyapatite (HA) and other multimodal (MM) chromatographic systems. We will employ the tools of high-throughput screening (HTS) and molecular simulation to provide fundamental insight into the nature of this selective behavior and to address several critical issues related to the design of selective displacers for MM systems. A robotic high-throughput displacer screening technique will be employed to screen for selective displacers in MM systems and to evaluate the effects of protein, displacer structure, mobile phase characteristics and displacer concentration. This work will employ protein and displacer libraries previously developed in our group to establish model protein pairs and potential displacers for further analysis. Multicomponent adsorption isotherms will also be generated to study competitive binding, synergistic interactions and hysteresis effects in these displacer-protein-MM systems. Column studies will be conducted to determine the level of enhanced selectivity and performance that can be achieved with this hybrid approach. A dynamic affinity plot analysis will also be carried out to examine if the enhanced selectivity and displacement-desorption transitions can be understood from a mass action perspective. A wide array of experimental and theoretical techniques will be employed to examine the interactions of selective displacers with immobilized MM ligands. Surface plasmon resonance spectroscopy will examine kinetic effects while isothermal titration calorimetry will provide insights into the underlying mechanisms involved in the selective displacement process in MM systems. A series of molecular simulations will be carried out to study the interactions of selective displacers with MM surfaces and to provide fundamental insight into the importance of various intermolecular interactions. Molecular simulations of multicomponent adsorption will also be carried out with small proteins in solutions of displacers and salt counter-ions to obtain further insights into competitive binding and the mechanism of the displacement process in MM systems. Finally, the use of selective displacers for addressing challenging biopharmaceutical separations in MM systems will be evaluated using complex biological mixtures provided by an industrial collaborator. There is an urgent need to develop new technologies to meet the growing challenge for safe, low cost next generation biotherapeutics ranging from proteins (e.g., antibodies, enzymes) to polysaccharides (e.g., heparin, hyaluronic acid) to cellular based technologies. The proposed project will produce a powerful new hybrid separations technology to address this need. The insights obtained in the proposed research will aid in the development and implementation of improved selective displacement MM chromatographic systems for the purification of therapeutic proteins. This will potentially eliminate the need for expensive affinity chromatographic systems and will enable a reduction in the number of required downstream processing steps, with a significant impact on the costs of producing biopharmaceuticals. The fundamental studies described in this project will also provide important insights into modification of hydrophobicity/philicity and binding in complex multicomponent mixtures which will establish a new platform for deeper understanding of a range of systems where these effects are important (e.g. bioseparations, biosensors, biomaterials). Clearly, if successful the proposal will have a significant and broad impact on bioprocessing and a number of health related fields. The proposed research will also have an important impact on the education of both graduate and undergraduate chemical engineering students as well as high school students. The Cramer laboratory has a long track record of producing chemical engineers with a first rate training in chromatographic bioprocessing. This training will continue with the students involved in this project. The proposed research will be carried out in the Center for Biotechnology and Interdisciplinary Studies which provides excellent multidisciplinary training opportunities for students and exposure to a wide range of experimental and simulation techniques. The research developed in this project will also be incorporated into a chemical engineering senior laboratory chromatography experiment recently developed the PI as well as a course on Chromatographic Separation Processes. Finally, simulations performed in this project, as well as conceptual parts of molecular interactions will motivate new aspects of the Molecularium project, which uses animation movies to teach and inspire students at all levels about the fascinating world of molecules.