Endocrine Abstracts (2019) 65 P79 | DOI: 10.1530/endoabs.65.P79

Development of a long-acting parathyroid hormone for the treatment of hypoparathyroidism

Faez Alshehri, Richard Ross & Ian Wilkinson

University of Sheffield, Sheffield, UK

Background: Parathyroid hormone (PTH) is a peptide hormone consisting of 84 amino acids with residues 1–34 responsible for its biological activity. Hypoparathyroidism is a rare, complex condition with a patient predisposition toward impaired mineral homeostasis. Replacement with Natpara (PTH 1-84) requires daily injections and is still complicated by fluctuating calcium levels. An unmet need exists for a long-acting treatment that is effective. It is hypothesised that a PTH-Fusion with growth hormone binding protein (GHBP) will retain biological activity and have reduced renal clearance and proteolysis, thus prolonging the half-life of PTH.

Methods: Stable clones of two PTH fusions were generated: 14A8 (PTH fused to extracellular domain of PTH receptor and GHBP) and 14A7 (PTH fused to GHBP). Correct gene integration was confirmed by RT-PCR and sequencing. Proteins were expressed from suspension adapted CHO stable cell lines in roller bottle culture. 14A8 was purified by ion exchange and affinity chromatography. Potency assays were completed using a Dual Luciferase Reporter Assay (DLRA) and the PTH responsive cell line UMR-106.

Results: Sequencing confirmed gene integration and protein expression was confirmed by western blotting for both stable clones. 14A8 was purified to 2 mg/ml from roller bottle culture and shown to separate between 75 and 100 kDa by SDS–PAGE. Using the DLRA an EC50 for both PTH 1-34 and 14A8 were obtained: mean 54 vs. 1214 nM. 14A8 was ˜22-fold less potent but had a higher maximal fold induction of 2.4-fold compared to PTH 1-34.

Conclusion: Fusions were successfully expressed from a stable CHO cell line and preliminary purification of 14A8 achieved. 14A8 exhibited a higher maximal fold induction, but was less potent than PTH 1-34. The reason for this is presently unknown but could be due to increased stability offered by the fusion, and will be further investigated.

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