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Generation of a long acting GCSF

alshehri, Abdulrahman (2016) Generation of a long acting GCSF. PhD thesis, University of Sheffield.

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Abstract

Rationale: Current therapies require daily injections of GCSF to treat patients with neutropenia and response to treatment is often unpredictable as GCSF is rapidly cleared. A number of approaches to reducing GCSF clearance have been tried mainly through conjugation with another moiety. The technologies already being employed, include PEGylation, immunoglobulins or albumin to increase the half‐life of GCSF. However, although these approaches have reduced clearance the pharmacokinetic profile of GCSF has remained unpredictable. Aim and Hypothesis: a glycosylated linker between two ligands could delay clearance with out blocking bioactivity. Methodology: GCSF tandem molecules with linkers containing between 2-8 N-linked glycosylation sites (NAT motif) and their respective controls (Q replaces N in the sequence motif NAT so there is no glycosylation) were cloned, and sequenced. Following expression in CHO cells, expressed protein was quantified by ELISA and analysed by western blot to confirm molecular weights and protein integrity. In vitro bioactivity was tested using an AML-193 proliferation assay. IMAC was used to purify the protein. Pharmacokinetic and pharmacodynamic of GCSF tandems were measured in Sprague Dawley rats. Results: Purified glycosylated tandem molecules showed increased molecular weight according to the number glycosylation of their sites when analysed by SDS-­PAGE. All GCSF tandems showed increased in vitro bioactivity in comparison to rhGCSF. GCSF2NAT, GCSF4NAT and GCSF8NAT containing 2, 4 & 8 glycosylation sites respectively and GCSF8QAT displayed a three fold increased terminal half‐life compared to that published for GCSF, however there was no difference in serum half-­‐life according to the level of glycosylation. Both GCSF2NAT and GCSF4NAT showed a higher increase in the percentage of neutrophils over controls at 12 hrs post injection only. In contrast, GCSF8NAT exhibited a higher increase in neutrophil levels over controls at 48 hrs. Conclusion: Using glycosylated linkers in GCSF tandems results in molecules with increased molecular weight according to the number of glycosylation sites. Tandems of GCSF have increased in vitro bioactivity compared to monomeric GCSF. Tandems with and without glycosylation had three fold greater half-­lives than rhGCSF. There was evidence that GCSF8NAT was biologically active in vivo. The results confirm the hypothesis that it is possible to predictably increase the molecular weight of GCSF tandems and retain biological activity but this was not associated with a predictable prolongation of the serum half‐life.

Item Type: Thesis (PhD)
Academic Units: The University of Sheffield > Faculty of Medicine, Dentistry and Health (Sheffield) > Medicine (Sheffield)
Identification Number/EthosID: uk.bl.ethos.682311
Depositing User: Dr Abdulrahman alshehri
Date Deposited: 30 Mar 2016 10:25
Last Modified: 12 Oct 2018 09:25
URI: http://etheses.whiterose.ac.uk/id/eprint/12274

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