Characterization of binding between model protein GA-Z and human serum albumin using asymmetrical flow field-flow fractionation and small angle X-ray scattering.

Jaeyeong Choi,Jonas Fransson,Ann Terry,Vilhelm Ek,Christopher A G Söderberg,Lars Nilsson,Ulla Elofsson,Marie Wahlgren

doi:10.1371/journal.pone.0242605

Abstract

Protein-based drugs often require targeted drug delivery for optimal therapy. A successful strategy to increase the circulation time of the protein in the blood is to link the therapeutic protein with an albumin-binding domain. In this work, we characterized such a protein-based drug, GA-Z. Using asymmetrical flow field-flow fractionation coupled with multi-angle light scattering (AF4-MALS) we investigated the GA-Z monomer-dimer equilibrium as well as the molar binding ratio of GA-Z to HSA. Using small angle X-ray scattering, we studied the structure of GA-Z as well as the complex between GA-Z and HSA. The results show that GA-Z is predominantly dimeric in solution at pH 7 and that it binds to monomeric as well as dimeric HSA. Furthermore, GA-Z binds to HSA both as a monomer and a dimer, and thus, it can be expected to stay bound also upon dilution following injection in the blood stream. The results from SAXS and binding studies indicate that the GA-Z dimer is formed between two target domains (Z-domains). The results also indicate that the binding of GA-Z to HSA does not affect the ratio between HSA dimers and monomers, and that no higher order oligomers of the complex are seen other than those containing dimers of GA-Z and dimers of HSA.

Highlights

For many biological drugs, a short half-life of the drug circulating in the blood is a problem
We found two peaks around the retention times of Human serum albumin (HSA), but with small tails appearing at GA-Z/ HSA mole ratio of 0.14
Searching the protein data bank (PDB) we found a crystal structure of HSA bound to a GA-domain (PDB ID: 1TF0)

Summary

Introduction

A short half-life of the drug circulating in the blood is a problem. Short half-life will either require frequent dosing or the need for high doses and, in the worst case, can mean that the drug is not efficient enough to reach the market. All of these scenarios cause problems for patients, e.g. low patient compliance, increased need for high drug doses which leads to increased risks for adverse side effects, and a risk for low efficacy. The latter could result in no viable treatment for a disease. Strategies to increase blood circulation time for biological drugs are needed, and one such strategy is albumin binding

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