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ABSTRACT
Developing protein therapeutics has posed challenges due to short circulating times and toxicities. Recent advances using poly(ethylene) glycol (PEG) conjugation have improved their performance. A PEG-conjugated hemoglobin (Hb), Hemospan, is in clinical trials as an oxygen therapeutic. Solutions of PEG-hemoglobin with two (P5K2) or six to seven strands of 5-kD PEG (P5K6) were studied by small-angle x-ray scattering. PEGylation elongates the dimensions (Hb < P5K2 < P5K6) and leaves the tertiary hemoglobin structure unchanged but compacts its quaternary structure. The major part of the PEG chains visualized by ab initio reconstruction protrudes away from hemoglobin, whereas the rest interacts with the protein. PEGylation introduces intermolecular repulsion, increasing with conjugated PEG amount. These results demonstrate how PEG surface shielding and intermolecular repulsion may prolong intravascular retention and lack of reactivity of PEG-Hb, possibly by inhibiting binding to the macrophage CD163 hemoglobin-scavenger receptor. The proposed methodology for assessment of low-resolution structures and interactions is a powerful means for rational design of PEGylated therapeutic agents.
INTRODUCTION
Poly(eihylene) glycol (PEG) is an inert, synthetic polymer increasingly being used to modify drugs and proteins to enhance stability and retention time and lower toxicity (1,2). For example, covalent attachments of PEG to the surfaces of acetylcholinesterase or Interferon-β-1b increase their circulatory residency times (3,4). In parallel, PEG-conjugated hemoglobin (Hb) is being explored for use as a hemoglobinbased oxygen carrier (HBOC). Hemospan is human oxyHb conjugated to an average of six to seven PEGs (5-kD) per Hb tetramer. This product, also referred to in the literature as MP4, is formulated at ~4 g/dl and has been the subject of biochemical (5-8) and physiologic investigations that have shown it to be a safe and effective oxygen-transport agent in animal models of hemodilution (9,10) and hemorrhage (11,12). Hemospan has successfully completed Phase 1(13) and Phase II clinical trials (14), with Phase III trials underway in Europe.
Despite ongoing development of several therapeutic PEG-conjugated proteins, including Hb, the effect of the PEG polymer on the three-dimensional (3D) structure of proteins is poorly understood. The relatively large, highly flexible PEG-polymer conjugates impede crystallization and structural determination by protein crystallography. Thus, considering the substantial amount of biochemical and structural data on Hb, the corresponding PEG-conjugated Hb (PEG-Hb) provides an excellent model to study the general...