Many of the benefits of PEGylated proteins lie in the properties of PEG. PEG is inert, non-toxic, and non-immunogenic.11 The polymer is easily cleared from the body, through the kidney for molecules with molecular weight below 20 kDa, or through
the liver for molecules with molecular weight above 20 kDa.12 PEG is a viscous liquid at molecular weights lower than 1000 and solid at higher molecular weights. PEG is typically prepared
by anionic polymerization, providing a variety of molecular sizes. It contains two OH- groups that can be activated, 13 and the polymerization can be controlled such that the molecular weight distribution is narrow. Currently, there are two
main types of commercially available PEG: linear and branched.
PEG -(CH2-CH2-O)n -, is a highly soluble, amphiphilic polyether diol. Its solutions are neutral. X-ray structural analysis shows that PEG chains
can assume two extreme structural conformations: a zigzag, random coil structure for shorter chains, or a winding, helical
structure for longer chains.14 These conformations are reversible in water and dependent on solution conditions.15 The presence of the ether oxygen atom allows for the hydration of the highly mobile chains through the formation of the
oxonium ion. Thus, in aqueous solutions PEG can adopt a structure with helical elements in which three water bridges are formed
per monomer unit.16 Consequently, the highly mobile PEG chains are heavily hydrated, and have a large exclusion volume that can inhibit the
approach of another molecule. For this reason, PEG's hydration properties determine the overall hydrodynamic properties of
Consequently, the most important property of a PEGylated protein is the increased molecular size resulting from the large
hydrodynamic volume of the PEG. The effective volume of PEG has a more significant effect on proteins that are smaller than
70 kDa,6 as described in the following explanation.
A protein's size may be approximated by estimating the hydrodynamic radius RH using two equations derived for proteins from pulsed-field gradient nuclear magnetic resonance (NMR) studies.17 In the equation for a folded protein,
RH native = 4.75 N0.29 
N represents the number of protein residues. According to Equation 1, the RH value of myoglobin, a protein of medium size is about 20 Å. A value of 17 Å for the radius of gyration, Rg, has been determined by other methods.18 The calculated RH is in agreement with the value of 22 Å measured by a diffusion experiment for lactalbumin, a protein similar to myoglobin
in terms of structure, molecular weight, and number of amino acids.
For polymer chains in solvents, the size description is based on statistical conformations of the chain in two dimensions.
The dynamic scaling is described by the gyration radius Rg as a function of its monomer unit number N0 :19
Rg ~ N0
For a 20-kDa PEG chain (454 monomer units), Equation 2 yields a Rg value of 98 Å. A value of 70 Å for the Rg gyration radius of a 20-kDa PEG has been previously reported.20 Using a random coil configuration for PEG, the Flory radius estimates that a 20-kDa PEG occupies a sphere with a radius
of 76.5 Å.21 When comparing a 20-kDa PEG with a native protein of medium molecular weight (~150 amino acids), a ratio of ~5 for the polymer
gyration radius to protein hydrodynamic radius is obtained. Because of the polymer's hydration, the three-dimensional hydrophilic
environment that PEG creates around the protein is likely much larger. Clearly, at the macromolecular level the PEG 's properties