PEGylation of Proteins: A Structural Approach

Structural properties of PEGylated proteins could play an increasingly important role in developing optimal therapeutic protein drugs.
Apr 01, 2006
Volume 19, Issue 4

A. Sorina Morar
With the completion of the Human Genome Project, molecular biologists are continuously discovering new classes of proteins and new therapeutic uses for known proteins. The ~30,000 genes defined by the Human Genome Project translate into 300,000 to one million proteins.1 Converting these proteins into effective biopharmaceuticals, however, may require challenging formulation development, because most recombinant proteins have limited chemical or physical stability in liquid state. For this reason, a renewed interest in conjugating proteins with polyethylene glycol (PEG) can be expected, with the final goal of bringing PEGylated protein drugs to market.


Depending on their ionic charge, size, and structure, proteins vary in their thermal stability, solubility, and susceptibility to proteolysis. The intermolecular packing and surface chemistry of proteins determine many of these biopharmaceutical properties, and many stress factors can cause protein unfolding and degradation, ultimately leading to loss of biological activity.2 Slight changes in pH, ionic strength, or temperature, for example, all can reduce biological activity in vivo. 3 Other potential stress factors include proteases and oxidation. In vivo, these protein properties can translate into a high clearance rate of a therapeutic protein from the body. In addition, short plasma half-life and reactions with the immune system complicate effective delivery of therapeutic proteins in humans.4 Bypassing these problems can be accomplished by either stabilizing the proteins or increasing their solubility, thus allowing for low dosage volumes and longer circulation times.

To achieve the desired stability and solubility, proteins can be modified using methods such as crosslinking,5 fusion to other proteins, changing the oligomerization state, glycosylation, mutations of cysteine residues, or polymer attachment.6 Currently, one of the current most successful methods for stabilizing proteins and increasing their solubility is to use polymer therapeutics, i.e., to link an active molecule to a polymer molecule such as polyethylene glycol (PEG). Polymer therapeutics includes polymer drugs, polymer conjugates, and polymeric micelles.7 In general, conjugating a protein to a polymer can accomplish several desirable objectives: a longer in vivo half-life; reduced immunogenicity, toxicity, and clearance rate through the kidneys; successful transportation across a cell membrane; protection against proteolysis; modification of electro-osmotic flow; increased pH and thermal stability; a low volume of distribution and sustained adsorption from the injection site; and improved formulation properties of the protein. These superior properties can increase effective potency8 , improve response to the drug, increase patient tolerance and reduce side effects, reduce overall dosage, decrease office visits, and lower the cost to the patient. A better drug profile and an improved quality of life are thus achieved.9 In the biotechnology industry, better biophysical characterization and understanding of PEGylated protein properties would allow for better control of the conjugation reaction and an improved drug product.


PEG is FDA approved for human administration by mouth, injection, or dermal application. It has been used for many years in various capacities. Early work used PEG in crystallography for crystal growth, purification in two-phase systems, incorporation into liposomes for an increased serum lifetime, or attachment to surfaces to reduce protein adsorption.10 In addition, PEG has been used to promote correct protein folding.

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