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

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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.