Competitive Ion-Exchange Adsorption of Proteins

University Of Houston, Houston TX

Investigators

Abstract

Abstract CTS-0004544 Competitive Ion-Exchange Adsorption of Proteins Richard Willson University of Houston This study applies a unique set of test proteins, which are identical in size and shape but which differ in color and adsorption behavior, for use as probes of competitive adsorption phenomena. These proteins are derived from the recombinant soluble core of cytochrome b5, expressed in E. coli from a synthetic gene. The adsorption behavior of these proteins is tailored by conservative mutations of surface glutamic-acid and aspartic-acid residues. The work explores the competitive equilibrium adsorption behavior of mixtures of these proteins of similar size but differing adsorption affinities. Nonporous adsorbents are used for the initial equilibrium work to avoid complications from dynamical/transport effects which are investigated separately. A shallow-bed, rapid-filtration technique is used to observe adsorption, desorption, and displacement dynamics on a short time scale. Specific topics being addressed include the effects of competitors on adsorption heterogeneity and adsorbent selectivity among proteins. Recombinant pharmaceutical proteins now make a substantial contribution to health care. Examples include insulin for treatment of diabetes, tPA for unblocking coronary arteries in heart attacks, and new, effective medicines for anemia, hemophilia, and breast cancer. The production of recombinant proteins generally consists of two stages, cultivation of bacteria or mammalian cells and then purification of the desired product from the complex mixture produced by the cells. Protein purification is the more expensive stage and is the subject of intense research interest and commercial development. This work addresses the fundamental behavior underlying the performance of the most widely-used purification method, selective adsorption of contaminants onto charged particles in ion-exchange chromatography. The particular focus is the competitive adsorption of proteins - how the presence of contaminants can alter the adsorption behavior of a desired product. This competition among proteins, which is essential to separating the mixture, is relatively little-investigated and poorly understood. Better understanding of these mechanisms will lead to better separation technologies and thus to higher-quality, lower-cost medical products.