Endocrine Abstracts

Rationale: Over the last 20 years, granulocyte colony-stimulating factor (GCSF) has become a recognized therapy in the treatment of patients suffering from neutropenia. Current therapies require daily injections of GCSF to stimulate stem cell production 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, included PEGylation, immunoglobulins and glycosylation to increase the half-life of rhG-CSF, however although these approaches have reduced clearance the pharmacokinetic profile has remained unpredictable. The aim of this project is to create a long acting GCSF with predictable pharmacokinetic profile to provide a more effective treatment for generating stem cells for bone marrow transplantation.

Hypothesis: The incorporation of variable glycosylated linkers between two GCSF molecules will create a construct with high molecular weight and protected from proteolysis resulting in reduced clearance while retaining bioactivity.

Methodology: GCSF tandem molecules with linkers containing between two and eight NAT glycosylation motifs and their respective controls (Q replaces N in the sequence motif NAT) were cloned, and sequenced. Following expression in Chinese hamster ovary (CHO) cells, expressed protein was quantified by ELISA and analysed by SDS–PAGE to confirm molecular weights. In vitro bioactivity was tested using an AML-193 proliferation assay. Immobilised metal affinity chromatography (IMAC) was used to purify the protein.

Results: GCSF tandem molecules with a flexible (Gly₄Ser)_n linker and glycosylation sites were expressed in CHO cells. The degree of glycosylation correlated with an increase in molecular weight, with the addition of 2, 4 and 8 glycosylation motifs, reaching a peak MW of around 52, 60 and 70 kDa compared to 45.4, 45.4 and 48.5 kDa for non-glycosylated controls respectively. Bioactivity was confirmed for all glycosylated tandems and un-glycosylated controls comparable to native GCSF.

Conclusion: These results demonstrate that the use of glycosylated linkers to generate protein-tandems results in glycosylation and an increased molecular weight whilst retaining bioactivity. Future studies are required to investigate stability and in vivo studies to test protein clearance.