PEGylation of Proteins: A Structural Approach - Structural properties of PEGylated proteins could play an increasingly important role in developing optimal therapeutic protein drugs. - BioPharm

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PEGylation of Proteins: A Structural Approach
Structural properties of PEGylated proteins could play an increasingly important role in developing optimal therapeutic protein drugs.


BioPharm International
Volume 19, Issue 4

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


Figure 1. Protein PEGylation results in a complex mixture of mono-, di-, and multi-PEGylated species
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:

PEG + P → PEG–P

PEG-P + PEG → (PEG)2 –P

(PEG)2 -P + nPEG → (PEG)n+2 –P


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