Silica colloidal crystals as emerging materials for the high-throughput sizing of proteins by packed capillary electrophoresis.

摘要

Protein heterogeneity is currently one of the leading problems in formulation of therapeutic proteins. The effectiveness of therapeutic proteins is diminished when they exhibit heterogeneity due to aggregates and degradation by-products. Furthermore, guidelines set forth by the FDA require therapeutic proteins to have high stability as well as high purity. With development time on the order of years and costs incurred in the millions, pharmaceutical companies are investing more time in finding fast and efficient ways to screen for protein heterogeneity. Current methods such as native polyacrylamide gel electrophoresis (PAGE), size-exclusion chromatography (SEC) and capillary electrophoresis (CE) are time consuming, require large amount of samples, and are not always easily implementable with instrumentation. Our approach to these issues involves the use of silica colloidal crystals bearing brush layers of polyacrylamide. Using established correlations between molecular weight and reduced mobility, selectivity is compared in silica colloidal crystals and gels. Due to low broadening effects in colloidal crystals, fast sieving separations of proteins have been observed in capillaries over distances as short as 6.5 mm in 72 seconds with exceptionally low plate heights. When adapted to a 96 well plate format, throughput can be increased a 100 fold. Our work explores the feasibility of high throughput characterization of protein aggregates and degradation products using a clinical antibody donated by the Eli Lilly Corporation.;Chapter 4 and 5 cover a project related to the investigation of the structural backbone of asphaltenes, which are a cause of serious concern to the petroleum industry. They have many undesirable effects such as precipitating in the pipelines and some of their metal content will foul catalysts used in oil processing. There are several endeavors dedicated to elucidating their structures. The one our group undertook involved synthesizing smaller asphaltene compounds that retained some of the key structural features associated with real asphaltenes. Fragmentation studies of these model compounds using tandem mass spectrometry revealed an unexpected fragmentation pattern which is meant to be used in elucidating the structure of real asphaltenes compounds.;In Chapter 6, work toward the synthesis of the photoaffinity labeled analog of Callipeltin A is presented. Capilleltin A is a natural product with desirable anti-HIV activity. Unfortunately, not much is known about its mode of action with its therapeutic targets. In order to learn more, we decided to replace the hydroxyl group of one of the non-proteogenic amino acid residues by a highly reactive group such as azide. From past studies, such a replacement is not expected to affect the bioactivity of Callipeltin A. A beta-MeOTyr azide analog was synthesized and fully characterized as part of the progress made.