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的替代范例来改善复杂生物样品的分离：使用混合模式这些材料被装入不锈钢管中，当与传统的液体流动相梯度结合时，固定相上的化学相互作用位点的强度会沿管的长度变化，从而按顺序置换分析物分子。力量随着流动相溶剂的增加，此类材料有可能在大生物分子的分离中提供显着提高的选择性。在实践中实现这种改进的选择性的一个重要障碍是在受控的条件下通过实验制造这些混合模式梯度固定相的能力。目前的工作将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，将实验设计和模拟相结合来解决这一挑战。科学发现和学生培训这项研究预计将推进分离科学领域的知识，特别是在以分析大型生物相关分子（包括蛋白质、肽和最终的单克隆抗体）为目标的领域，这些分子对新型生物制剂的开发特别感兴趣。它探索了一个原创概念，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、色谱柱改造等方面的宝贵专业知识。同时，他们将成为 LC 和生物分子混合物分离方面的专家，使其在大多数化学分离领域具有独特的用途。另一种范式将梯度置于固定相上，最近的模拟工作表明，当与流动相梯度结合时，双固定相梯度可以“打开以前从未见过的”。在目前的工作中，将解决与适合生物分子分离的混合模式固定相梯度的制造和实施相关的挑战，并探索新的方法。以梯度方式用官能化一氯硅烷（例如苯基、C8、C4...）策略性地修改颗粒填充硅胶柱。梯度的陡度将发生变化和优化，以便当与流动相梯度协同选择性和/或谱带压缩相结合，将在生物分子分离中观察到模拟方法，以预测色谱响应并优化获得最佳色谱柱所需的梯度长度和组成。分离将通过模拟与实验相结合来实现，从而避免了材料开发中通常使用的试错方法。从长远来看，通过这项研究开发的新色谱柱技术和伴随的模拟有可能影响这些领域。蛋白质组学和脂质组学，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智力价值和更广泛的影响审查标准进行评估，被认为值得支持。