Improving the LC Separation of Biomolecule Mixtures using Novel Mixed-Mode Gradient Stationary Phases

在化学系化学测量和成像项目的支持下，弗吉尼亚联邦大学的Maryanne Collinson教授和她的团队正在寻求通过探索分离科学中的另一种范式来改善复杂生物样品的分离：使用混合模式梯度固定相。 将这些材料装入不锈钢管中，固定相上的化学相互作用位点的强度沿管的长度沿着变化。当与传统的液体移动的相梯度（其随着移动的相溶剂的强度增加而顺序地置换分析物分子）结合时，这种材料具有在分离大生物分子中提供显著改善的选择性的潜力。在实践中实现这种改进的选择性的一个重要障碍是以受控和可预测的方式实验性地制造这些混合模式梯度固定相的能力。 这一挑战将在目前的工作中通过实验设计和模拟相结合，通过与色谱模拟及其预测能力领域的专家Sarah Rutan博士的持续合作来解决。 该项目将为科学发现和学生培训和学习提供机会。 这项研究预计将推动分离科学领域的知识，特别是在目标分析大型生物相关分子的领域，包括蛋白质，肽，以及最终对新型生物药物开发特别感兴趣的单克隆抗体。该项目探索了一种新颖的概念，旨在提高分离的选择性，并最终提高复杂蛋白质样品中化学相似分析物的分辨率。参与该项目的学生将获得色谱柱填充，硅烷化学改性二氧化硅以及使用TGA和其他光谱和显微镜工具进行详细表征的宝贵专业知识。同时，他们将成为LC和分离生物分子混合物的专家，使他们在生物制药领域具有独特的应用价值。在大多数化学分离中，梯度在移动的相中。 另一种范例将梯度置于固定相上。 最近的模拟工作表明，双固定相梯度与移动的相梯度结合时，可以“打开以前看不见的选择性”在大的生物分子的分离。 在目前的工作中，与制造和实施这种混合模式的固定相梯度适合于生物分子分离的挑战将得到解决。 通过这项工作的过程，将探索新的方法来战略性地用官能化的一氯硅烷（例如，苯基、C8、C4、.）以梯度的方式。 梯度的陡度将被改变和优化，使得当与移动的相梯度结合时，在生物分子的分离中将观察到协同选择性和/或谱带压缩。 将开发模拟方法来预测色谱响应并优化色谱柱制造条件。获得最佳分离所需的梯度长度和成分将通过将模拟与实验相结合来获得，从而避免了材料开发中通常使用的试错法。从长远来看，通过这项研究开发的新柱技术和伴随的模拟有可能影响蛋白质组学和脂质组学领域，以及二维液相色谱分离。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

With support from the Chemical Measurement and Imaging Program in the Division of Chemistry, Professor Maryanne Collinson and her group at Virginia Commonwealth University are seeking to improve the separation of complex biological samples by exploring an alternate paradigm in separation science: the use of mixed-mode gradient stationary phases. These materials are packed into stainless steel tubes and the strength of the chemical interaction sites on the stationary phase is varied along the length of the tube. When coupled with a traditional liquid mobile phase gradient, which displaces the analyte molecules sequentially as the strength of mobile phase solvent is increased, such materials have the potential to provide significantly improved selectivity in the separation of large biomolecules. An important obstacle to realizing such improved selectivity in practice is the ability to experimentally fabricate these mixed-mode gradient stationary phases in a controlled and predictable fashion. This challenge will be addressed in the present work through a combination of experimental design and simulation via a continued collaboration with Dr. Sarah Rutan, an expert in the field of chromatographic simulations and their predictive power. This will project provide opportunities for both scientific discovery and student training and learning. This research is expected to advance knowledge in the field of separation science, particularly in areas that target the analysis of large, biologically relevant molecules including proteins, peptides, and ultimately monoclonal antibodies that are of particular interest in the development of novel biologic pharmaceuticals. It explores an original concept aimed to improve the selectivity of a separation and ultimately the resolution of chemically similar analytes within complex protein samples.Students involved in this project will obtain valuable expertise in the packing of chromatography columns, the modification of silica using silane chemistry, and its detailed characterization using TGA and other spectroscopic and microscopic tools. Simultaneously, they will become experts in LC and the separation of mixtures of biomolecules making them uniquely employable in the biopharmaceutical field. In most chemical separations, the gradient is in the mobile phase. An alternate paradigm puts the gradient on the stationary phase. Recent simulated work has shown that dual stationary phase gradients when coupled with a mobile phase gradient can “open up previously unseen selectivities” in the separation of large biomolecules. In the present work, the challenges associated with the fabrication and implementation of such mixed-mode stationary phase gradients suitable for biomolecule separations will be addressed. Through the course of this work, new approaches will be explored to strategically modify a particle-packed silica column with a functionalized monochlorosilane (e.g., phenyl, C8, C4,...) in a gradient fashion. The steepness of the gradients will be varied and optimized so that when coupled with a mobile phase gradient synergistic selectivity and/or band compression will be observed in the separation of biomolecules. Simulation methods will be developed to predict the chromatographic response and to optimize the conditions for column fabrication. The gradient lengths and compositions needed to obtain optimum separations will be obtained by coupling simulations with experiments, thus avoiding the trial-and-error approach usually used in materials development. Over the long term, the new column technology and accompanying simulations developed through this research have the potential to impact the fields of proteomics and lipidomics, as well as two-dimensional liquid chromatographic separations.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.