Development of New Separation Methods for Personalized Medicine

Alpha1-acid glycoprotein (AGP) plays an important role as a carrier protein for basic and neutral pharmaceuticals in the circulatory system. The interaction of those ligands with AGP affects its pharmacological effect and the active concentration of pharmaceuticals that are available for the target tissue. The binding between drug and AGP is susceptible to change due to the variation of both AGP concentration and compositions of glycoforms and variants under disease states. This dissertation described the development of various analytical separation-based methods to characterize various attributes of AGP for sample both in a purified form and in serum. The first and second chapters provided an introduction for high performance affinity chromatography (HPAC), capillary electrophoresis (CE) and especially their applications in the analysis of biological, pharmaceutical and clinical samples. The third and fourth chapters described the development of CE methods for the analysis of charge variants of AGP. The separation conditions were optimized such as capillary coating methods, pH and the use of buffer additives. This allowed a reproducible separation of charge variants for AGP as a purified sample. Electrophoretic injection and sample pretreatments were optimized and used for the charge variant analysis of AGP from serum. The established method relied on the integrated injection technique that allowed purification of AGP from serum and sensitivity enhancement through sample stacking. The fifth and sixth chapters described the development of high-performance lectin affinity microcolumns, their applications in the glycoform analysis of AGP and two-dimensional separations. Silica-based lectin microcolumns were prepared and optimized using concanavalin A (Con A) and Aleuria Aurantia Lectin (AAL) as affinity stationary phases. The established microcolumns can separate glycoforms of AGP based on the degree of branching and fucosylation of N-glycans. The sixth chapter explored the off-line coupling of different modes of affinity chromatography with each other or with CE for the two-dimensional separations of AGP. The seventh chapter described the development of a chromatographic one-site immunometric assay for measuring AGP level in serum. The established assay can provide a sensitive measurement of AGP level from serum sample in a high throughput manner