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


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 typical reaction set-up places all the PEGylation components in the same solution phase. The PEG-to-protein ratio usually ranges from 1:1 to 5:1, at a pH dictated by the desired specificity of the reaction.

When optimizing a PEGylation reaction toward a high yield and purity for the desired species, the formulator must consider the PEGylation reagent used, reaction conditions, and purification issues.34 The most important factors that affect the PEGylation reaction include, but are not limited to, protein concentration, PEG-to-protein ratio, reaction pH and temperature, reaction time, protein characteristics (molecular weight, surface area, polarity, local amino acid conditions at the PEGylation site, such as lysine pKa, and site accessibility).30 Other aspects of the reaction that could be critical to the reaction outcome are mixing and the PEG addition rate, and the presence of hydrophobic or hydrophilic co-solutes and buffer components. Cosolvents can alter solution properties such as ionic strength, viscosity, and dielectric properties, or can perturb the conformational distribution of PEGylated species. Examples of such co-solutes are unreactive PEGs or dextrans, sugars, salt, alcohol, or detergents. In theory, by fine tuning of these parameters the reaction can be optimized to achieve a high yield of the desired PEG conjugate.


Figure 2. The PEGylation reaction for a model protein and PEG. The equilibrium between worm- and shell- like conformations would include typical random fluctuations of an unstructured polymer loop until it finds either a surface or its other end. In the spherical conformation, some degree of loop formation in surrounding water is acceptable. While covalently bound, the PEG may also interact non-specifically with the protein.
Although not much is known about the structural properties of the protein conjugate, it is generally agreed that the dominant properties of PEG play a large role in the conjugate's overall properties. Because PEG can adopt various conformations dependent upon solution conditions,14 multiple conformations of the PEG-protein hybrid can exist, most likely stabilized by an intricate H-bond lattice. In one conformation of the protein-polymer conjugate, water solvates hydrophilic regions around the protein while hydrophobic PEG clusters interact with corresponding protein patches. These reactions create a shell-like structure (PEGshell-Protein in Figure 2) in which PEG is wrapped around the surface of the protein. Physiologically, this structure translates into a higher stability and reduces the immune system's recognition of the protein.3,35 There is the alternate model, however, in which there is no PEG-protein interaction. The conjugate forms a worm-like helical structure (PEGworm-Protein in Figure 2) in which PEG fluctuates freely in solution. The first model could explain how, at the macromolecular level, PEG coupling to protein can effectively mask the protein surface from proteolytic cleavage. In both models the physical properties of PEG dominate, generating the non-immunogenic benefits in the polymer-protein hybrid.

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