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Next-Generation Affinity Chromatography with PEGylated Ligands

使用聚乙二醇化配体的新一代亲和色谱法

基本信息

批准号：
1159886
负责人：
Todd Przybycien
金额：
$ 30万
依托单位：
Carnegie-Mellon University
依托单位国家：
美国
项目类别：
Standard Grant
财政年份：
2012
资助国家：
美国
起止时间：
2012-08-01 至 2016-07-31
项目状态：
已结题

来源：
https://www.nsf.gov/awardsearch/showAward?AWD_ID=1159886&HistoricalAwards=false
关键词：
Next Generation Affinity Chromatography PEGylated

项目摘要

PrzybycienCBET 1159886This NSF award by the Chemical and Biological Separations program supports work by Professor Todd M. Przybycien to develop new separations technology for use in the biotechnology industry and to develop a corresponding laboratory and teaching module for secondary school chemistry students as an introduction both to separations technology and to the biotechnology industry. The separations technology development effort involves the modification of affinity chromatography media, commonly used in the production of pharmaceutical proteins such as monoclonal antibodies, so that it is more resistant to fouling by contaminant species, thereby increasing manufacturing efficiency. The secondary education module development effort involves the creation and deployment of a set of laboratory experiments and supporting lecture materials in affinity partitioning technology that can be used to augment current high school chemistry curricular materials in the area of the separation of matter by demonstrating the ideas of affinity and specificity in separation operations. These educational materials will also be used to provide exposure to the biotechnology industry, a key domestic industrial sector, as the experiments are motivated by and have applications to bioprocessing and bioseparations.Affinity chromatography is a premier bioseparations technology in which specific binding interactions are exploited to achieve selective recovery of a target biological macromolecule from a complex biological mixture. Monoclonal antibodies, or MAbs, are the dominant class of biologic products from the biopharmaceutical industry. Affinity chromatography using media with immobilized MAb-specific binding groups such as Stapylococcal Protein A, or SPA, is typically the central unit operation around which generic, or platform, MAb purification processes are developed. Our specific research approach is to modify SPA-based affinity chromatography media by chemically attaching poly(ethylene glycol), or "PEG", an inert polymer, to the SPA binding group. This chemical modification, or PEGylation, will improve media selectivity by decreasing non-specific binding interactions with SPA and will improve media robustness by increasing the stability/degradation resistance of SPA. That this approach should be successful is supported by the well-known use of PEGylation to impart stealthy characteristics to protein-based drugs without adversely affecting biological binding activity, to enhance the physical stability of proteins, and to improve the fouling resistance of surfaces towards proteins and other biologically-derived species. PEGylation strategies (size, extent, structure of attached PEG chains; masking of MAb-specific binding sites on SPA to preserve binding affinity) will be evaluated with respect to their impact on important chromatographic performance metrics including MAb binding capacity and MAb binding specificity in the presence of customary contaminants (CHO host cell protein, DNA and virus) and during many cycles of repeated use. If successful, a new class of high-performance affinity chromatography media based on ligand PEGylation will result.The proposed research aims to invoke a step-change in the selectivity and robustness of affinity chromatography media, and SPA-based media in particular, to enable a corresponding step-change improvement in chromatographic performance. PEGylated affinity chromatography media with increased specificity and robustness relative to current media can reduce process development time for new biological pharmaceuticals (improved specificity translates into fewer downstream purification steps to develop and validate) and can increase process throughput for new and existing biological pharmaceuticals (improved specificity translates into shorter, less-stringent wash and clean-in-place cycles and fewer downstream steps to operate; improved robustness translates into greater binding capacity retention, less ligand leaching and ability to use ligands with preferred specificity but decreased stability, and more operating cycles before media replacement). Greater speed-to-market and process throughput, particularly for the critical MAb class of biopharmaceuticals, can drive down treatment costs. The proposed research will comprise the research training of a Ph.D. student and several undergraduate engineering students, poising each for career opportunities in the domestic biopharmaceutical industry.

化学和生物分离项目授予的NSF奖项支持了托德M.与Przybycien合作开发用于生物技术工业的新分离技术，并为中学化学学生开发相应的实验室和教学模块，作为分离技术和生物技术工业的介绍。分离技术开发工作涉及亲和色谱介质的改性，其通常用于生产药物蛋白质如单克隆抗体，使得其更能抵抗污染物的污染，从而提高生产效率。中等教育模块的开发工作涉及创建和部署一套实验室实验和支持讲座材料的亲和分区技术，可用于增强目前的高中化学课程材料在该地区的物质分离，通过展示分离操作中的亲和性和特异性的想法。这些教育材料也将被用来提供接触生物技术工业，一个关键的国内工业部门，因为实验的动机和应用于生物加工和生物分离。亲和色谱是一种主要的生物分离技术，其中利用特异性结合相互作用，实现从复杂的生物混合物中选择性回收目标生物大分子。单克隆抗体，或MAb，是生物制药工业中占主导地位的一类生物制品。使用具有固定化的MAb特异性结合基团（例如葡萄球菌蛋白A或SPA）的介质的亲和层析通常是开发通用或平台MAb纯化方法的中心单元操作。我们的具体研究方法是通过化学连接聚（乙二醇），或“PEG”，一种惰性聚合物，SPA结合基团，以修改SPA为基础的亲和色谱介质。这种化学修饰或PEG化将通过减少与SPA的非特异性结合相互作用来提高培养基选择性，并将通过增加SPA的稳定性/抗降解性来提高培养基耐用性。这种方法应该是成功的，这得到了众所周知的聚乙二醇化的使用的支持，以赋予基于蛋白质的药物隐形特性，而不会不利地影响生物结合活性，以增强蛋白质的物理稳定性，并改善表面对蛋白质和其他生物衍生物质的耐污染性。将评价PEG化策略（连接的PEG链的大小、程度和结构;掩蔽SPA上的MAb特异性结合位点以保持结合亲和力）对重要色谱性能指标的影响，包括在存在常规污染物（CHO宿主细胞蛋白、DNA和病毒）的情况下以及在多次重复使用循环期间的MAb结合能力和MAb结合特异性。如果成功的话，一类新的基于配体PEG化的高性能亲和层析介质将产生。拟议的研究旨在调用亲和层析介质的选择性和鲁棒性的阶跃变化，特别是基于SPA的介质，以实现相应的阶跃变化改善色谱性能。相对于当前介质具有增加的特异性和稳健性的PEG化亲和层析介质可以减少新生物药物的工艺开发时间（改进的特异性转化为更少的下游纯化步骤来开发和验证）并且可以增加新的和现有的生物制药的工艺通量（改进的特异性转化为更短的、不太严格的清洗和就地清洁循环以及更少的下游操作步骤;改进的稳健性转化为更大的结合能力保留、更少的配体浸出和使用具有优选特异性但稳定性降低的配体的能力，以及在培养基更换之前更多的操作循环）。更快的上市速度和工艺生产量，特别是对于关键的单克隆抗体类生物制药，可以降低治疗成本。拟议的研究将包括一名博士的研究培训。学生和几个本科工程专业的学生，平衡每个在国内生物制药行业的就业机会。