Competitive Ion-Exchange Adsorption of Proteins

摘要：cts -0004544蛋白质的竞争性离子交换吸附本研究采用一组独特的测试蛋白，它们在大小和形状上相同，但在颜色和吸附行为上不同，作为竞争性吸附现象的探针。这些蛋白来源于细胞色素b5的重组可溶性核心，在大肠杆菌中通过合成基因表达。这些蛋白质的吸附行为是由表面谷氨酸和天冬氨酸残基的保守突变定制的。这项工作探讨了这些蛋白质的大小相似，但不同的吸附亲和混合物的竞争平衡吸附行为。无孔吸附剂用于初始平衡功，以避免动力学/输运效应的复杂性，这是单独研究的。采用浅床快速过滤技术在短时间尺度上观察吸附、解吸和驱替动力学。讨论的具体主题包括竞争者对蛋白质间吸附不均一性和吸附剂选择性的影响。重组药物蛋白现在对卫生保健作出了重大贡献。例如用于治疗糖尿病的胰岛素，用于疏通心脏病发作时冠状动脉阻塞的tPA，以及用于治疗贫血、血友病和乳腺癌的新型有效药物。重组蛋白的生产通常包括两个阶段，细菌或哺乳动物细胞的培养，然后从细胞产生的复杂混合物中纯化所需产品。蛋白质纯化是成本较高的阶段，是研究兴趣和商业开发的主题。这项工作解决了最广泛使用的净化方法的基本行为，在离子交换色谱中，污染物在带电粒子上的选择性吸附。特别关注的是蛋白质的竞争性吸附-污染物的存在如何改变所需产品的吸附行为。蛋白质之间的竞争对分离混合物至关重要，但研究相对较少，了解也很少。更好地了解这些机制将带来更好的分离技术，从而产生更高质量、更低成本的医疗产品。

AbstractCTS-0004544Competitive Ion-Exchange Adsorption of ProteinsRichard WillsonUniversity of HoustonThis 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.