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Ruben G. Carbonell Frank Hawkins Kenan Distinguished Professor Chemical and Biomolecular Engineering North Carolina State University Identification and Characterization of Small Ligands for Affinity Separations of Biological Molecules |
Abstract
The biotechnology industry is under increased pressure to cut production costs and to improve the characterization and safety of pharmaceutical products. As a result, there is a great deal of interest in the use of affinity chromatography to reduce the number of steps in the isolation and purification of biological molecules. Even though affinity chromatography is used extensively on a small scale for protein purification and characterization, its use for larger scale industrial processes is quite limited. The most specific and most common ligands used on a small scale are antibodies, which can be produced from animal and cell sources for just about any target. Unfortunately, the cost of producing pure antibodies on a large scale for industrial applications is prohibitive. In addition, harsh product elution and wash conditions tend to denature the antibodies, and leakage from the column can result in dangerous immunological responses in patients.
For these reasons, a great deal of effort is being placed on the search for alternative ligands that are less costly and more robust than antibodies to facilitate the introduction of affinity chromatography in large-scale applications. This paper describes the use of combinatorial libraries in identifying small peptide ligands for affinity adsorption. These libraries contain millions of compounds and are synthesized directly on chromatographic resin to ensure that any ligands found can bind the target molecule in the same particle format to be used in columns. Sensitive detection methods have been developed for screening these libraries for the best ligands, and detailed characterizations of the binding properties have been carried out for many protein-ligand pairs. Even though these small ligands are not as specific as antibodies, they are often specific enough to be able to isolate or concentrate a target protein from a complex mixture. Since the association constants are weaker than antibodies, elution conditions are not as strong. The peptides used are normally hexamers that do not require a tertiary structure for their specificity and can be synthesized chemically on a large scale under GMP conditions. As a result, these ligands are less costly and more robust than antibodies.
To illustrate the major features of this technology, we will discuss some recent results on the identification and characterization of a small peptide ligand that binds to Staphylococcal enterotoxin B (SEB) for detection and removal applications. This peptide is able to recognize and separate SEB from a complex mixture of E-coli cell homogenate, and can recognize the structural differences between denatured or ‘nicked' SEB and native SEB. Chromatographic characterization of the peptide indicates that the there is a significant adsorption rate limitation on the separation. The chromatographic parameters were used in the design of a column to reduce the concentration of this toxin by several orders of magnitude. In addition, we will present some new results on a small peptide that is able to bind specifically to the Fc fragment of human immunoglobulin G (HIgG). This peptide is the only small ligand ever reported to share the ability of Protein A to recognize only the Fc fragment of HIgG. The peptide can bind several isotypes of IgG but shows little binding to other types of immunoglobulins. This peptide has the potential of serving as an alternative to Protein A and G in HIgG purification.
Biography
Ruben G. Carbonell is the Frank Hawkins Kenan Distinguished Professor of Chemical and Biomolecular Engineering at North Carolina State University . He joined NC State in 1984, after ten years in the Chemical Engineering Department at the University of California , Davis . He was Department Head of Chemical Engineering at NC State from 1994 to 1999. He is currently Director of the William R. Kenan, Jr. Institute for Engineering, Technology, & Science, and Co-Director of the NSF Science and Technology Center for Environmentally Responsible Solvents and Processes. His main areas of research include the development of novel high-pressure carbon dioxide based processes, and of new separation and detection methods for biological molecules using small ligands identified from combinatorial libraries. He is author and co-author of over 170 publications in technical journals and 25 patents in a wide range of subject areas. Professor Carbonell earned his BS degree in chemical engineering from Manhattan College in 1969 and the PhD degree in chemical engineering from Princeton University in 1973. He is currently on the Scientific Advisory Boards of Prometic LifeSciences, Inc., Micell Technologies, Inc. and Pathogen Removal and Diagnostic Technologies, Inc. He was named NC State University Alumni Distinguished Graduate Professor in 1994 and won the NC State University Alumni Outstanding Research Award in 1989. He also won the Maurice Simpson Technical Editor's Award for Excellence in the Field of Contamination Control from the Institute of Environmental Sciences in 1992 and the R. J. Reynolds Award for Excellence in Teaching, Research and Extension from the NC State College of Engineering in 1990. In 2001 he also received the Alcoa Outstanding Research Award from the College of Engineering . In 2003 he was named a Fellow of the American Institute of Chemical Engineers.