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.
STRUCTURAL CONFORMATION OF PEGYLATED PROTEIN SPECIES
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.
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.