Recent advances in the development of therapeutic biomolecules derived from living organisms, or biologics, have lead to their widespread applications in the treatment of disease. From monoclonal antibodies to recombinant proteins, hundreds of biologics are now in the market pipeline. As the use of such protein-based treatments gains traction, the requirements for purifying active biologics from complex mixtures becomes more pressing. A major challenge in translating bench-scale proteomics research to commercial-scale drug manufacturing is the purification process. The ability to isolate only the protein of interest as well as the speed by which this can be accomplished are important factors in increasing therapeutic efficacy and reducing biologic manufacturing costs. This project seeks to enable the efficient fractionation of proteins from complex mixtures using membrane- based separations technology. Membrane-based fractionation enables large volumes of material to be filtered over short periods of time. This project optimizes both physical and chemical characteristics of membranes to achieve very selective and high throughput protein purification. One key to achieving rapid, high quality protein purifications using membrane technology is the appropriate tailoring of membrane structure and chemistry. Terapore's ultrafiltration membranes are made from a unique, self-assembling organic molecule that creates a high density of very uniform pores. The high pore density results in high membrane permeability, enabling large volumes to be processed with relatively small membrane areas, while the uniform pore sizes enable similarly size molecules to be efficiently isolated based on size- exclusion principles. In this project, the critical issue for enhancing the membrane selectivity beyond structural considerations through chemically tuning the membrane surface and pore wall chemistry will be addressed. In particular, this project explores how surface customization of the membrane structure will result in the ability to distinguish between similarly sized proteins based on the chemistry of the target protein. Specific goals in the project include the quantitative functionalization of membrane surfaces using both terminal and cross-linking agents, which will be monitored using nuclear magnetic resonance spectroscopy and Fourier-transform infrared spectroscopy. The functionalized membrane will be evaluated for permeability and compared with throughput for untreated membranes using a dead-end stirred cell filtration setup. The membranes will also be challenged both single-protein and multi-protein buffered feed solutions to evaluate rejection characteristics under various solution processing conditions. Feed and permeate protein concentrations will be monitored using UV-vis spectroscopy and gel permeation chromatography, and corroborated using high-resolution HPLC analyses. Finally, the membranes will be challenged with fermentation broth feed solutions to determine performance against industrial feed streams.
Public Health Relevance Statement:
Public Health Relevance: High purity protein samples are important for both bench-scale research and large-scale industrial applications, however, processing technologies for achieving high purity are cumbersome and expensive. Membrane-based protein purification offers a promising route to simplifying biomolecule separation processes. This project focuses on enabling the rapid and selective purification of therapeutic proteins by combining novel and structurally ideal ultrafiltration membranes with electrostatically active surfaces.
Project Terms:
Address; Area; base; bioprocess; Bovine Serum Albumin; Buffers; Cell Membrane Permeability; Cells; Characteristics; Charge; Chemicals; Chemistry; commercialization; Complex Mixtures; Confidential Information; copolymer; cost; crosslink; Data; density; Disease; Drug Formulations; Electrostatics; Equation; Exclusion; feeding; Fermentation; Filtration; Fourier Transform; Fractionation; Gel Chromatography; Goals; Hemoglobin; High Pressure Liquid Chromatography; improved; infrared spectroscopy; interest; Isoelectric Point; Lead; Life; Marketing; Measures; Membrane; Methodology; Monitor; Monoclonal Antibodies; Myoglobin; NMR Spectroscopy; novel; Organism; Performance; Permeability; Pharmaceutical Preparations; Procedures; Process; protein complex; protein purification; Proteins; Proteomics; public health relevance; Publishing; Reaction Time; Recombinant Proteins; Reporting; Research; research study; Resolution; Route; Sampling; solute; Solutions; Spectroscopy, Fourier Transform Infrared; Spectrum Analysis; Speed (motion); Stream; Structure; Surface; Technology; Temperature; Testing; Text; Therapeutic; therapeutic development; therapeutic protein; Time; Traction; Translating; Treatment Efficacy; Ultrafiltration; Work
