Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE credits
Glycoproteins are proteins with glycans located at the protein surface. These glycans are composed of monosaccharides covalently bound to each other in branced or linear structures. Sialic acids are monosaccharides that are located at terminal positions on the glycans and have been suggested to be of great importance for the proteins function and communication with surrounding cells and other proteins. Sialic acids have been show nto affect the glycoproteins half-life and activity. Furthermore, small modifications of sialic acids can affect the efficacy ad the interaction capacity of glycoproteins. Reseach has shown that it is possible to study sialic acids and their effects on glycoproteins. Furthermore, almost half of all the plasma proteins are glycoproteins, and most likely have sialic acids. It is therefore opportune to look into glycoproteins as drug candidates. Regulators as FDA and EMA already have demands for characterization of sialic acids in drug development. Moreover, the sialic acid content can be used as control for product consistency between batches. A pharmaceutical company needs to lie ahead and in accordance with the regulations, and to do so today it is essential to put resources into developing reliable methods for sialic acid analysis.
This report suggest an analytical proceduret o analyze the sialic acid content of glycoproteins by using two model glycoproteins, bovine fetuin and apo-transferrin bovine. Different hydrolysis methods for sialic acids release from glycoproteins, bovine fetuin and apo-transferrin bovine. Different hydrolysis methods for sialic acids release from glycoproteins are optimized and compared to find the superior hydrolysis method. The released sialic acid content is identified and quantified usign HPAE-PAD, which is a liquid chromatographic technique at high pH with electrochemical detection.
Acetic acid hydrolysis is show nto be superior to hydrochloric acid hydrolysis in releasing sialic acid from the model glycoproteins due to less genereation of hydrolysis byproducts. The analytical procedure described for sialic acids released by acetic acid hydrolysis from fetuin is also qualified considering specificity, linearity, accuracy and precision and was found scientificlly sound.