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

使用新型混合模式梯度固定相改善生物分子混合物的液相色谱分离

• 批准号: 2305102
• 负责人: Maryanne Collinson
• 金额: $ 48万
• 依托单位: Virginia Commonwealth University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2026-07-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2305102&HistoricalAwards=false
• 关键词: Improving; LC; Separation; Biomolecule; Mixtures

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.

在化学划分的化学测量和成像计划的支持下，玛丽安·科林森（Maryanne Collinson）教授及其小组在弗吉尼亚联邦大学（Virginia Commonwealth University）寻求通过探索分离科学中的替代范式来改善复杂生物样品的分离：使用混合模式梯度固定阶段。这些材料被装入不锈钢管中，固定相位的化学相互作用位点的强度沿着管的长度变化。当与传统的液体流动相位梯度结合时，随着流动相偿付能力的强度提高，该分子分子依次置换分析物分子时，这种材料具有在大型生物分子的分离中显着提高选择性的潜力。实践中实现这种改善的选择性的重要障碍是能够以受控且可预测的方式实验实验制造这些混合模式梯度固定相。通过与色谱模拟领域的专家Sarah Rutan博士的持续合作，通过实验设计和模拟的结合，将在演讲工作中解决这一挑战。这将为科学发现以及学生培训和学习提供机会。预计这项研究将促进分离科学领域的知识，特别是针对分析大型生物学相关分子在内的领域，包括蛋白质，胡椒粉和最终对新型生物学药物开发特别感兴趣的单克隆抗体。它探讨了一种原始概念，旨在提高分离的选择性，并最终在复杂的蛋白质样品中分辨出化学相似的分析物。该项目所涉及的学生将在色谱柱的包装，使用Silane化学修改及其使用TGA和其他光学光学和其他光学分析和其他光学学上的详细表征来获得有价值的专业知识。同时，它们将成为LC的专家，并将生物分子的混合物分离，从而使它们在生物制药领域中独特地使用。在大多数化学分离中，梯度处于流动阶段。另一种范式使梯度处于固定阶段。最近的模拟工作表明，当与流动相位梯度结合时，双重固定相梯度可以在大型生物分子的分离中“打开以前看不见的选择性”。在目前的工作中，将解决与适合生物分子分离的这种混合模式固定相梯度相关的挑战。通过这项工作，将探索新的方法，以策略性地修改粒子包装的二氧化硅色谱柱，其功能化的单氯硅烷（例如，苯基，C8，C4，C4，...）以梯度方式进行修改。梯度的钢性将被变化和优化，因此，在与流动相梯度的协同选择性和/或带压缩的相结合时，将在生物分子的分离中观察到。将开发仿真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组成将通过将模拟与实验耦合，从而避免通常用于材料开发中使用的试验方法。从长远来看，通过这项研究开发的新专栏技术和参与模拟有可能影响蛋白质组学和脂质组学领域，以及二维液体色谱分离。该奖项反映了NSF的法定任务，并通过使用该基金会的知识分子和更广泛的影响来评估CRITERIA CRITERIA。