Next-Generation Affinity Chromatography with PEGylated Ligands

PrzybycienCBET 1159886这项由美国国家科学基金会化学和生物分离项目颁发的奖项，支持Todd M. Przybycien教授开发用于生物技术行业的新分离技术，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行业的介绍。分离技术的开发工作涉及到亲和层析介质的修饰，通常用于生产药物蛋白质，如单克隆抗体，使其更能抵抗污染物的污染，从而提高制造效率。中学教育模块的开发工作包括创建和部署一套亲和分割技术的实验室实验和支持讲座材料，这些材料可以通过展示分离操作中的亲和和特异性的概念来增强当前高中化学课程材料中物质分离领域的内容。这些教育材料还将用于使人们了解生物技术工业，这是一个关键的国内工业部门，因为这些实验是由生物加工和生物分离推动的，并适用于这些实验。亲和色谱是一种重要的生物分离技术，利用特定的结合相互作用，从复杂的生物混合物中实现目标生物大分子的选择性回收。单克隆抗体或单克隆抗体是生物制药行业的主要生物产品。亲和层析使用固定单克隆抗体特异性结合基团（如葡萄球菌蛋白A或SPA）的介质，通常是开发通用或平台单克隆抗体纯化工艺的中心单元操作。我们的具体研究方法是通过将惰性聚合物聚乙二醇（PEG）化学连接到SPA结合基团上来修饰SPA基亲和层析介质。这种化学修饰或聚乙二醇化将通过减少与SPA的非特异性结合相互作用来提高介质选择性，并通过增加SPA的稳定性/抗降解性来提高介质稳健性。众所周知，聚乙二醇化可以赋予基于蛋白质的药物隐形特性，而不会对生物结合活性产生不利影响，增强蛋白质的物理稳定性，并提高表面对蛋白质和其他生物衍生物种的抗污染能力，这一方法应该是成功的。PEG修饰策略（附着PEG链的大小、范围、结构；在SPA上屏蔽MAb特异性结合位点以保持结合亲和力）将被评估其对重要色谱性能指标的影响，包括在常规污染物（CHO宿主细胞蛋白、DNA和病毒）存在和多次重复使用过程中，MAb结合能力和MAb结合特异性。如果成功，将产生一类基于配体聚乙二醇化的新型高性能亲和色谱介质。提出的研究旨在引起亲和色谱介质，特别是基于spa的介质的选择性和稳健性的阶梯式变化，从而使色谱性能得到相应的阶梯式改善。与现有介质相比，聚乙二醇化亲和色谱介质具有更高的特异性和稳健性，可以减少新生物药物的工艺开发时间（改进的特异性意味着需要开发和验证的下游纯化步骤更少），并可以增加新生物药物和现有生物药物的工艺吞吐量（改进的特异性意味着更短、更不严格的清洗和原位清洁周期，以及更少的下游操作步骤）；增强的鲁棒性意味着更大的结合能力保留，更少的配体浸出，以及使用具有首选特异性的配体的能力，但稳定性降低，以及在介质更换之前更多的操作周期)。更快的上市速度和流程吞吐量，特别是对于关键的单抗类生物制药，可以降低治疗成本。拟议的研究将包括一名博士生和几名工科本科生的研究培训，为每个人在国内生物制药行业的职业发展做好准备。

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.