UNS:Thermal control of avidity for separation of biologicals

1511227北卡罗来纳州U亲和层析利用固定在表面的分子或配体与目标物种之间的高度特异性相互作用。在拟议的工作中，将使用相同的合成方案与所提议的分子或配体一起生成亲和吸附剂，用于小规模和大规模分离。这些亲和吸附剂有望在灭菌后坚固耐用，并可多次重复使用。然而，预计用于一次性使用的高价值生物制剂的吸附剂的生产仍将具有成本效益。将与北卡罗来纳州的一家公司合作，探索开发用于复杂生物制剂的商用膜分离系统。为了确定温度敏感的亲和配体，将评估一种随着温度变化而改变构象的多肽支架。将产生两个模型靶点的亲和配体：表达在造血干细胞上的CD117和线粒体蛋白Tom22。随后，将在细胞和线粒体分离的背景下评估这些配体的有效性。因此，将筛选多肽的组合文库以确定模型靶标的多肽结合子。温度响应性配体也将使用产生的多肽来构建。随后，将评估温度对这些配体结合亲和力的影响。最后，将在分离CD117+细胞和线粒体的背景下评估用所选配体功能化的磁珠的使用。

1511227RaoNorth Carolina State UAffinity chromatography exploits the highly specific interaction between molecules or ligands immobilized on a surface and the target species of interest. In the proposed work affinity adsorbents will be generated with the proposed molecules or ligands for both small and large scale separations using the same synthesis scheme. These affinity adsorbents are expected to be robust and amenable to multiple re-uses after sterilization. Yet, it is expected that production of adsorbents for single-use applications with high value biologicals will still be cost-effective. Collaboration with a North Carolina State-based company will be undertaken to explore the development of commercial membrane-based separation systems for complex biologicals. To identify temperature-sensitive affinity ligands, a peptide-based scaffold that changes conformation in response to changes in temperature will be evaluated. Affinity ligands for two model targets will be generated: CD117, which is expressed on hematopoietic stem cells, and TOM22, a mitochondrial protein. Subsequently, the effectiveness of these ligands will be evaluated in the context of cell and mitochondrial separation. Accordingly, combinatorial libraries of peptides will be screened to identify peptides binders for the model targets. Thermo-responsive ligands will also be constructed using the peptides generated. Subsequently, the effect of temperature on the binding affinity of these ligands will be evaluated. Finally, the use of magnetic beads functionalized with the selected ligands will be evaluated in the context of separation of CD117+ cells and mitochondria.