A recent study shows that the viscosity radius of the PEGylated protein depends only on the molecular weight of the native
protein and the total weight of grafted PEG and suggests that PEG forms a dynamic layer over the surface of the protein.22
Not only does the protein size change upon PEGylation, but the conformation and electrostatic binding properties of a PEG-conjugated
protein might be different from those of the unmodified protein. When a PEG moiety is added to a protein, the resulting species is a polymer–protein hybrid, that does not necessarily retain
the physical properties of the protein. The conjugate's properties are dominated by sterical hindrance at and away from the
active site, conformational changes of both PEG and protein, altered binding properties resulting from changes in local pI,
pKa, hydrophobicity and hydrophilicity of the protein. Little is known about the effect of free or bound PEG on protein structure.
Most studies point to an unchanged protein secondary structure.23-25 As a result, most of the protein's biological activity is preserved.
In general, the pharmacokinetics and pharmacodynamic properties of a PEG-protein conjugate depends on the site at which the
PEG is attached, the molecular weight of the PEG used, the number of PEG molecules attached to a protein, and the stability
of the protein-PEG bond.26 For example, the size and structure of PEG largely determine the rate of clearance. The type of reactive moiety controls
the site and stability of the covalent link and also the number of PEGylation sites on a given protein. In addition, the PEG's
size and structure likely affect the maximum number of PEGylation sites that can be "accessed." Not surprisingly, the location
of the site attachment has been correlated with the pharmokinetic activity9 and stability of the PEGylated species in vivo as demonstrated by resistance to proteolytic degradation.27
THE PEGYLATION REACTION
The PEGylation reaction is defined as the covalent attachment of one or more PEG molecules to a biologically active protein.28 To optimize a PEGylation reaction, a formulation scientist must consider many factors, including the goal of PEGylation.
Typically, a minimal number of PEGylation sites is desired to reduce loss in bioactivity. PEG structure and size are just
two factors that can limit the degree of PEGylation. For example, branched PEGs increase the molecular weight of the mono-PEGylated
protein, and also can limit the steric availability of PEGylation sites. With a linear PEG, one expects to see a large gyration
radius as a result of increased chain length versus branched PEG of a similar molecular weight. Also, the interaction of a
branched PEG with protein could be different than that of a linear PEG. Thus, a variety of species can be synthesized.
In a typical reaction, an activated monofunctional PEG is reacted with one or more accessible lysine residues or the N-terminal
amino group. PEGylation at other nucleophilic sites such as cysteine, histidine, arginine or tyrosine also are possible.29-32 Depending on the activated form of PEG, an ester (PEG succinimidyl succinate) or urethane (PEG succinimidyl carbonate) covalent
bond is created between the polymer and the protein.33
In a PEGylation reaction, the protein solution is mixed with activated PEG. MonoPEGylated protein molecules with the most
reactive site are formed first, and then less reactive sites form diPEGylated species, and so on (Figure 1). These reactions
are consecutive pseudo first order reactions (under conditions of excess PEG), as described below:
Figure 1. Protein PEGylation results in a complex mixture of mono-, di-, and multi-PEGylated species
PEG + P → PEG–P
PEG-P + PEG → (PEG)2 –P
(PEG)2 -P + nPEG → (PEG)n+2 –P