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Affinity chromatographic studies of drug-protein binding in personalized medicine

Jeanethe Anguizola, University of Nebraska - Lincoln


In recent years there have been an increasing number of scientific advances dedicated to the transition from traditional medicine to a new era of data-driven personalized medicine. The customization of healthcare holds a great promise for providing faster diagnosis and more effective treatments for a variety of diseases and disorders, such as diabetes. The number of diabetic patients has increased at an alarming rate over the last 20 years, with an estimated 26 million children and adults in the United States suffering from this disease and related complications such as cardiovascular disease, kidney or liver damage, and blindness. Some of these complications have been associated with high levels of blood glucose and a process known as non-enzymatic glycation, which can produce structural and functional modifications of proteins in the human body. Among these proteins, human serum albumin (HSA) is an important transport protein for many drugs, hormones and fatty acids in the circulation. Therefore, a detailed discussion of the process of glycation on HSA is provided in this dissertation. One topic examined in this manuscript is the use of high-performance affinity chromatography (HPAC) and immobilized HSA microcolumns to examine the binding of various drugs to HSA as the glycation level is increased. This work was first performed by studying the binding of a number of sulfonylurea drugs to in vivo glycated HSA. Affinity microcolumns were prepared from only 20 μL of serum from individual patients with diabetes. The clinical samples were also analyzed using mass spectrometry to identify and quantify modifications of HSA that occur due to glycation. In addition, competition studies were used to investigate the effect of in vitro glycation and the presence of long chain fatty acids on the binding of drugs at the major binding sites of HSA. Frontal analysis and zonal elution experiments were also utilized to study the binding of drugs to alpha-acid glycoprotein (AGP), an acute phase protein. A novel on-column (in situ) protein entrapment approach was developed to prepare affinity microcolumns containing AGP. Lastly, the peak decay method and HPAC were used to examine the dissociation rate constants for several species of immunoglobulin G (IgG) from immobilized protein G supports. The approaches developed in this dissertation are not limited to glycated HSA and AGP but could be adapted to other modified proteins and disease states of interest to the scientific community and to the field of personalized medicine.

Subject Area

Chemistry|Analytical chemistry|Biochemistry|Pharmacy sciences

Recommended Citation

Anguizola, Jeanethe, "Affinity chromatographic studies of drug-protein binding in personalized medicine" (2013). ETD collection for University of Nebraska - Lincoln. AAI3590965.