Custom affinity chromatography : development of a novel platform for rapid creation and validation of affinity media using DNA aptamer based ligands

Biotechnology companies are now well skilled in the technologies and operations needed to manufacture biologic drugs safely for the treatment of major diseases. Those technologies have enabled development of highly efficient and cost-effective platforms for downstream processing of monoclonal antibody (mAb) based drugs. But translating those advances to create cost-effective DSP platforms for non-mAb protein therapeutics having relatively low annual production rates, often termed “orphan” drugs, has proven difficult. A key driver of the efficiency of mAb DSP platforms is the use of protein A affinity chromatography to capture and purify the product directly from clarified culture supernatants. Unfortunately, effective ligands for affinity capture of non-mAb biologics are generally not available. But this could change through the development of a technology that rapidly discovers and validates cost-effective affinity ligands against non-antibody protein targets. This project describes the development of a new technology pipeline to accelerate the discovery, optimization, and validation of affinity chromatography media that is specifically tailored to provide for robust economical capture of non-mAb biologic drugs from complex cell cultures. It is based on the use of DNA aptamers as affinity ligands discovered using an advanced aptamer screening technology we call High-Fidelity Systematic Evolution of Ligand by Exponential Enrichment (Hi-Fi SELEX). The refined, truly robust Hi-Fi SELEX technology described in this thesis greatly improves upon a proof-of-concept version of that method we recently described. This second-generation Hi-Fi SELEX method was used to successfully select high-affinity ligands against two non-mAb target proteins, human complement Factor D and human mesothelin. Anti-Factor D (aFD-30) aptamer against Factor D was then used as ligands in preparative affinity chromatography columns. The chemically modified aFD-30 with 3’ inverted dT nucleotide cap was immobilized on preparative affinity chromatography matrix for the capture and purification of Factor D from CHO cell supernatant. Standard column performance data were collected, including static and dynamic binding capacities, purities, concentration factors, and yields, which showed excellent separation performance. These results therefore demonstrate the potential of the proposed technology for custom design and validation of preparative chromatography media that can benefit the growing orphan drugs market by reducing manufacturing costs.Applied Science, Faculty ofChemical and Biological Engineering, Department ofGraduat