Antibody Purification via Affinity Chromatography that Utilizes the Unconventional Nucleotide Binding Site

1263713 Bilgicer本项目的目的是设计一种新的亲和层析技术，用于从细胞培养基和腹水中纯化单克隆和多克隆抗体。该方法靶向抗体上存在的非常规核苷酸结合位点（NBS），以促进其在亲和柱上的捕获。抗体对抗原具有非凡的特异性和亲和力，并用于包括检测、诊断、催化和治疗在内的大量应用中。 在过去的十五年中，治疗性抗体已经彻底改变了许多疾病的治疗，包括癌症和自身免疫性疾病。尽管对患者有益，但基于单克隆抗体的疗法价格昂贵，使许多患者负担不起。该成本的主要贡献者是在抗体生产期间使用蛋白A（或G）亲和柱。 这些柱是昂贵的并且具有短的循环寿命。该项目的结果可能导致低成本的纯化方法。 在初步研究中，PI用吲哚丁酸（IBA）靶向NBS。 初步结果表明，IBA柱对所选抗体的捕获率为95%，纯度为98%。PI的研究计划包括以下具体目标：i）筛选NBS的高亲和力小分子（核苷酸类似物）; ii）鉴定NBS树脂的最佳抗体纯化性能; iii）测定NBS柱达到的抗体产率和纯度; iv）测定柱再生和可重复使用性。 NBS柱的潜在优点是：i）降低生产成本，ii）增加柱容量，iii）增加柱寿命（更耐用的材料在苛刻的柱再生条件下是稳定的），iv）蛋白A/G不会浸出到纯化的抗体中，以及v）仅捕获和洗脱生物活性的纯化抗体。 由于核苷酸结合位点存在于所有抗体上，因此该柱可用于纯化来自所有物种的所有同种型的单克隆和多克隆抗体。更广泛的影响。 抗体在治疗、诊断和生命科学研究中具有重要意义。基于NBS的纯化方法将潜在地有助于提高质量以及降低与抗体生产相关的总成本，并且在所有这些领域都具有社会效益。除了拟议研究的科学影响外，PI还致力于增强教育氛围，以激励研究生，本科生和预科生追求科学和工程职业。作为一个初级教师，PI寻求利用该项目来帮助实现三个长期的教育目标：1）通过参与尖端研究来加强学生的本科教育。2)增加代表性不足的少数群体成员在科学领域的参与。PI已经招募了几名少数民族学生为他的研究小组3）提高地区高中的科学教育水平，培养少数民族学生？对科学和工程职业感兴趣。PI将帮助开发一个动手的职业决策研讨会，PI将在他的实验室每学期举办。 PI还寻求加强核心高中生物学。他与南本德新技术高中的教师合作，为一门新的生物技术课程设计课程。在南本德的新技术高中成功地建立了开创性的生物技术课程，可能会导致在全国新技术高中的实施。

1263713 BilgicerThe objective of this project is to engineer a novel affinity chromatography technique for purification of monoclonal and polyclonal antibodies from cell culture media and ascites fluids. This method targets the unconventional nucleotide binding site (NBS) that exists on antibodies to facilitate their capture on an affinity column. Antibodies have extraordinary specificity and affinity for antigens and are used in a vast array of applications including detection, diagnosis, catalysis, and therapy. In the last fifteen years, therapeutic antibodies have revolutionized the treatment of many diseases including cancers and autoimmune diseases. Despite their benefits to patients, monoclonal antibody-based therapies are expensive, making them unaffordable to many patients. A major contributor to this cost is use of Protein A (or G) affinity columns during antibody production. These columns are expensive and have a short cycle life. The results of this project may lead to a low-cost purification method. In preliminary studies, the PI targeted the NBS with indole butyric acid (IBA). Preliminary results showed that IBA column's capture efficiency for selected antibodies was 95%, with purity of 98%. The PI's research plan includes the following specific aims: i) screening for high affinity small molecules (nucleotide analog) for the NBS; ii) identification of the NBS resin for the best antibody purification performance; iii) determination of antibody yield and purity achieved by NBS column; iv) determination of column regeneration and reusability. The potential advantages of the NBS column are: i) reduced production cost, ii) increased column capacity, iii) increased column lifetime (more durable material is stable under harsh column regeneration conditions), iv) no leaching of protein A/G into purified antibody, and v) capture and elution of only bivalently active, purified antibody. Because the nucleotide binding site is present on all antibodies, the column can be used to purify both monoclonal and polyclonal antibodies of all isotypes from all species. Broader Impacts. Antibodies are of high significance in therapy, diagnosis and life-science research. An NBS-based purification method will potentially contribute to increasing quality as well as lowering the overall cost associated with antibody production and be of societal benefit in all these areas. In addition to the scientific impact of the proposed research, the PI is also committed to enhancing the educational climate to inspire graduate, undergraduate, and pre-collegiate students to pursue careers in science and engineering. As a junior faculty, the PI seeks to leverage the project to help achieve three long-standing goals in education: 1) Enhance the undergraduate education of students by participation in cutting edge research. 2) Increase the participation of members of underrepresented minorities in science. The PI has recruited several minority students for his research group 3) Improve the standard of science education in area high schools, and raise minority students? interest in careers in science and engineering. The PI will help develop a hands-on Career Decision Workshop that the PI will host in his laboratory each semester. The PI also seeks to enhance core-high school biology. He has teamed up with South Bend New Tech High School teachers to generate the curriculum for a new biotechnology course. Successful establishment of the pioneering biotechnology course in the New Tech high school in South Bend may lead to implementation in New Tech high schools throughout the nation.