Protein Separations Using Affinity Ultrafiltration with Small Charged Ligands

摘要项目名称：色谱生物处理中的蛋白质吸附和转运项目编号：cct -0345528项目负责人：Andrew zydney机构：宾夕法尼亚州立大学项目目的是研究一种利用小的、带电的、亲和配体的蛋白质纯化的新方法，通过亲和超滤实现高分辨率的分离。亲和超滤利用了分子识别可以获得的高选择性，但允许产物和配体在自由溶液中相互作用，从而消除了传质限制和对配体可及性的担忧，这些限制通常阻碍填料柱亲和色谱的应用。结合相互作用引起蛋白质-配体复合物的电荷变化，这被用于在超滤过程中利用静电相互作用获得高的产物保留率。最初的实验研究将使用Cibacron Blue作为带电亲和力配体，人类血清白蛋白作为模型蛋白。初步的理论计算表明，蛋白质电荷的变化将为无法结合Cibacron Blue的白蛋白和蛋白质杂质之间的膜分离提供高选择性。这些结果将证明该技术的可行性，同时为使用小电荷亲和配体的亲和超滤过程的设计和优化提供定量框架。就更广泛的影响而言，该项目将为研究生和本科生提供处理膜过程和蛋白质分离的实践经验。这项工作的结果可能有助于开发新技术，以合适的价格、规模和质量为高价值产品提供高分辨率蛋白质纯化。

AbstractProposal Title: Protein Adsorption and Transport in Chromatographic BioprocessingProposal Number: CTS-0345528Principal Investigator: Andrew ZydneyInstitution: Pennsylvania State UniversityThe objective of this project is to examine a novel approach for protein purification using small, charged, affinity ligands to achieve high-resolution separations by affinity ultrafiltration. Affinity ultrafiltration exploits the high selectivity that can be obtained by molecular recognition, but the product and ligand are allowed to interact in free solution, thereby eliminating the mass transfer limitations and concerns about ligand accessibility that often hinder applications of packed column affinity chromatography. The binding interaction causes a change in electrical charge of the protein-ligand complex, and this is used to obtain high product retention by exploiting electrostatic interactions during ultrafiltration. Initial experimental studies will be performed using Cibacron Blue as the charged affinity ligand and human serum albumin as the model protein. Preliminary theoretical calculations indicate that the change in protein charge will provide high selectivity for membrane separations between albumin and protein impurities that are unable to bind Cibacron Blue. These results will demonstrate the feasibility of this technique, while providing a quantitative framework for design and optimization of affinity ultrafiltration processes using small charged affinity ligands. In terms of the broader impacts, this project will provide graduate and undergraduate students with hands-on experience dealing with membrane processes and protein separations. The results of this work may aid in the development of new technologies that provide high-resolution protein purification at a price, scale, and quality appropriate for high-value products.