Identification of Binding Sites on Human Serum Albumin for Somapacitan, a Long-Acting Growth Hormone Derivative.

Eva Johansson,Christine B Schjødt,Jørgen Petersen,Jianhe Chen,Peter Thygesen,Helle Demuth,Tao Huang,Sanne Jensen,Anders D Nielsen,Charlotte Wiberg

doi:10.1021/acs.biochem.0c00019

Eva Johansson, Christine B Schjødt + Show 8 more

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https://doi.org/10.1021/acs.biochem.0c00019

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Abstract

Somapacitan, a human growth hormone derivative that binds reversibly to albumin, was investigated for human serum albumin (HSA) and HSA domain binding. Isothermal titration calorimetry (ITC) binding profiles showed high-affinity binding (∼100-1000 nM) of one somapacitan molecule and low-affinity binding (∼1000-10000 nM) of one to two somapacitan molecules to HSA. The high-affinity site was identified in HSA domain III using size exclusion chromatography (SEC) and ITC. SEC studies showed that the neonatal Fc receptor shields one binding site for somapacitan, indicating its position in domain III. A crystal structure of somapacitan in complex with HSA optimized for neonatal Fc receptor binding, having four amino acid residue replacements, identified a low-affinity site in fatty acid-binding site 6 (domain II). Surface plasmon resonance (SPR) showed these replacements affect the kinetics of the high-affinity binding site. Furthermore, small-angle X-ray scattering and SPR brace two somapacitan-binding sites on HSA.

Highlights

Somapacitan, a human growth hormone derivative that binds reversibly to albumin, was investigated for human serum albumin (HSA) and HSA domain binding
Somapacitan is a human growth hormone (GH) derivative that binds reversibly but extensively (>99%) to endogenous circulating albumin.[7]. It is in development for once-weekly treatment of GH deficiency (GHD) in children and adults and for short children born small for gestational age (SGA)
It was recombinantly produced in K. pastoris (P. pastoris) and purified by affinity chromatography, and bound fatty acid was removed by active charcoal treatment at low pH.[16]

Summary

Introduction

Somapacitan, a human growth hormone derivative that binds reversibly to albumin, was investigated for human serum albumin (HSA) and HSA domain binding. The long half-life of circulating albumin in human blood is a result of low renal clearance combined with pH-dependent endosomal rescue from degradation, mediated by the formation of a complex with the neonatal Fc receptor (FcRn)[1,2] (Figure 1a). The aim of this study is to further clarify the mechanism of action for extension of the somapacitan plasma half-life by identifying specific binding sites on HSA for somapacitan and elucidating the binding stoichiometry and the binding affinity for those sites. This is not a straightforward task as HSA has at least seven fatty acid-binding sites, named FA1−FA7 (Figure 1b). A combination of several different methods has been applied to illuminate intricacies of binding of somapacitan to HSA

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