Ultrahigh Performance Non-Denaturing Protein Chromatography Columns

项目总结 所建议的产品是一种分析规模的UHPLC柱，用于分离蛋白质药物 天然形式，第一阶段提案侧重于阴离子交换层析 分离机制。粘合相由多聚物链组成，在这种情况下是轴承 弱阴离子交换基团。这项研究将扩展到阳离子交换和疏水 相互作用层析在未来的第二阶段工作。这些非变性分离是 在蛋白质药物的开发中至关重要，因为它们允许分离的成分 在药理研究中用于药效、免疫原性和作用机制的检测。非- 蛋白质药物的变性色谱柱使用聚合物键合相，因为 这些都赋予了快速吸附和解吸所需的分子规模的灵活性。这个 非变性色谱柱的整体市场规模约为10亿美元， 市场领先者包括Thermo-Dionex。安捷伦、沃特斯和Sepax。当前存在的问题 柱子有两个：分色的分辨率相对较低，而柱子有 短暂的一生。初步结果表明，两种分辨率柱的寿命都可以通过 创新制造工艺。后者认为分辨率和寿命是有限的 因为聚合物是可压缩的，高压和环境之间的循环 通过常规使用柱的压力逐渐扰乱填料以降低 随着时间的推移，分离。建议的柱将首先紧密堆积成不可压缩的。 二氧化硅颗粒给出了一种恒压填料。然后聚合物粘合相将被 从二氧化硅颗粒表面的引发剂在柱内生长，形成均匀的 粘合阶段。拟议的工作有两个具体目标。第一个目标是开发一个 聚合键合相的柱内生长的自动化程序。第二个目标是 将栏目表现与领先的商业栏目在以下方面进行比较 具有已知电荷异质性和柱寿命的单抗的拆分。 这项工作的结果将确立将非变性产品线商业化的可行性。 蛋白质药物的色谱柱。这一概念将在未来的第二阶段推广。 研究解决制造中的放大问题并开发其他非变性 分离方法，包括阳离子层析和疏水作用层析。

PROJECT SUMMARY The proposed product is an analytical scale, UHPLC column for separating protein drugs in native form, with the Phase I proposal focused on anion exchange chromatography as the separation mechanism. The bonded phase is made of polymer chains, in this case bearing weak anion exchange groups. The study will be expanded to cation exchange and hydrophobic interaction chromatography in future Phase II work. These non-denaturing separations are essential in the development of protein drugs because they allow the separated components to be tested for efficacy, immunogenicity and mechanism in phamacological research. Non- denaturing chromatography columns for protein drugs use polymeric bonded phases because these impart the molecular-scale flexibility needed for fast adsorption and desorption. The overall market size for non-denaturing chromatography columns is on the order of $1B, and the market leaders include Thermo-Dionex. Agilent, Waters and Sepax. The problems with current columns are two-fold: the separations are relatively low in resolution and the columns have short lifetimes. Preliminary results show both resolution column lifetime can be improved by an innovative manufacturing process. The latter considers that resolution and lifetime are limited because polymers are compressible, and the cycling between high pressure and ambient pressure through routine use of the column progressively disturbs the packing to degrade the separation over time. The proposed columns will be made by first tightly packing incompressible silica particles to give a pressure-invariant packing. Then the polymer bonded phase will be grown inside the column from initiators on the surfaces of the silica particles, forming a uniform bonded phase. The proposed work entails two specific aims. The first aim is to develop an automated procedure for in-column growth of the polymeric bonded phase. The second aim is to compare the column performance with those of leading commercial columns with respect to resolution of monoclonal antibodies having known charge heterogeneity and column lifetime. The outcome of the work will establish the feasibility of commercializing a line of non-denaturing chromatography columns for protein drugs. The concept will be extended in the future Phase II research to address scale-up issues in manufacturing and to develop other non-denaturing separations, including cation chromatography and hydrophobic interaction chromatography.