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Title: Studies on the specificities of in vivo and in vitro bioassays used in the potency determination of therapeutic erythropoietin
Author: Liefooghe, Emily Claire
Awarding Body: Open University
Current Institution: Open University
Date of Award: 2006
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Erythropoietin (Epo), a kidney-produced cytokine, promotes the proliferation and differentiation of erythroid progenitors into mature red blood cells. The in vivo biological activity of Epo is dependent on it being adequately glycosylated. Glycosylation is a variable process and its precise effect on clearance mechanisms within the body has yet to be fully established. Studies have shown, however, that the loss of the terminal sialic acid residue, leading to exposure of the penultimate galactose residue, results in a dramatic reduction in activity, owing to the liver's galactose-dependent clearing action. Current in vitro cell-based bioassays for Epo are unaffected by such a phenomenon and therefore do not correlate with the invivo bioassays. As part of this project, strategies were devised for rendering cell-based bioassays sensitive to sialylation. The strategies' common feature was the inclusion of a mechanism for depleting or sequestering desialylated Epo via a galactose-binding protein (derived from either plants or the human liver). The assays were tested usinga range of Epo samples desialylated to various degrees by neuraminidase treatment. This provided proof of principle that rational manipulation of in vitro hioassays rendering them sensitive to a factor known to affect in vivo activity can allow the prediction of bioactivity without the use of live animals. Two Epo sample sets were investigated in addition to the neuraminidase-treated sample set - one produced by different culture and purification procedures, the other consisting of a batch of pharmaceutical-grade Epo separated into fractions by ion-exchange HPLC. Investigations were carried out by means of the most sensitive assay developed as well as a range of biological, biochemical and physico-chemical techniques with a view to producing an analytical and comparative study. The results suggested that the relationship between galactose exposure and in vivo biological activity might not be as straightforward as had been initially thought.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available