Protein Separations Using Affinity Ultrafiltration with Small Charged Ligands

摘要提案标题：蛋白质在色谱生物处理中的吸附和转运提案号：CTS-0345528主要研究者：Andrew Zaverney机构：宾夕法尼亚州立大学本项目的目的是研究一种新的蛋白质纯化方法，该方法使用小的、带电荷的亲和配体通过亲和超滤实现高分辨率分离。 亲和超滤利用了通过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，而这些限制和担忧通常会阻碍填充柱亲和层析的应用。结合相互作用引起蛋白质-配体复合物的电荷变化，并且这用于通过利用超滤期间的静电相互作用来获得高产物保留。 将使用汽巴蓝作为带电荷的亲和配体和人血清白蛋白作为模型蛋白进行初始实验研究。初步理论计算表明，蛋白质电荷的变化将为白蛋白和不能结合汽巴蓝的蛋白质杂质之间的膜分离提供高选择性。 这些结果将证明这种技术的可行性，同时提供了一个定量的框架设计和优化亲和超滤过程中使用小电荷的亲和配体。 在更广泛的影响方面，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，以适合高价值产品的价格，规模和质量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.