EXPERT REVIEW The Immunogenicity of Polyethylene Glycol: Facts and Fiction Huub Schellekens & Wim E. Hennink & Vera Brinks Received: 11 January 2013 / Accepted: 24 April 2013 / Published online: 15 May 2013 # Springer Science+Business Media New York 2013 ABSTRACT An increasing number of pegylated therapeutic proteins and drug targeting compounds are being introduced in the clinic. Pegylation is intended to increase circulation time and to reduce an immunogenic response. Recently however a number of publications have appeared claiming that the poly- ethylene glycol (PEG) moiety of these products in itself may be immunogenic and that the induced anti-PEG antibodies are linked to enhanced blood clearance and reduced efficacy of the products. A critical review of the literature shows that most, if not all assays for anti-PEG antibodies are flawed and lack specificity. Also the biological effects induced by anti-PEG anti- bodies lack the characteristics of a bona fide antibody reaction. Standardization of the anti-PEG assays and the development of reference sera are urgently needed. KEY WORDS immune assays . immunogenicity . pegylation . therapeutic proteins ABBREVIATIONS ABC Accelerated blood clearance BSA Bovine serum albumin ELISA Enzyme linked immunosorbent assay EMA European medicines agency FDA Food and drug administration Ig Immunoglobulin OVA Ovalbumin pDNA Plasmid DNA PEG Polyethylene glycol INTRODUCTION Pegylation is frequently used to improve the clinical prop- erties of therapeutic proteins (1,2). It increases the protein size, inhibits proteolysis and renal filtration and thereby improves the pharmacokinetics of the protein drug. In ad- dition, polyethylene glycol (PEG) has a simple structure and is chemically inert. It also has an excellent solubility and no effect on protein conformation. Pegylation is therefore con- sidered an effective method to improve protein stability and to decrease the immunogenic potential of the modified proteins. Pegylation is also used to improve drug delivery systems (3). The long circulating property of PEG coated liposomes as well as other colloidal nanoparticles has been generally attributed to suppression of protein adsorption onto their surfaces which renders them invisible (or Stealth) for mac- rophages. In addition PEG has very low toxicity and be- cause of its simple structure is assumed to be of low immunogenicity itself. Several pegylated products have been approved by the FDA, EMA and other regulatory authorities and are use clinically with success. These products include PegIntron®, a pegylated form of interferon-alpha 2b, Pegasys®, a pegylated form of interferon- alpha 2a, Neulasta®, a pegylated form of granulocyte colony stimulating factor and Mircera®, a pegylated epoetin-beta. Long-term treat- ment of hundreds of thousands of patients with these prod- ucts has confirmed their safety. This very positive clinical experience with marketed pegylated therapeutic proteins is in sharp contrast with an increasing number of reports claiming that PEG can be highly immunogenic, and might compromise efficacy and safety of protein drugs (4). Moreover there are reports suggesting an increasing incidence of anti-PEG antibodies in normal donors. It has been suggested that these anti-PEG H. Schellekens : W. E. Hennink : V. Brinks (*) Department of Pharmaceutics Utrecht Institute of Pharmaceutical Sciences Utrecht University, Universiteitsweg 99 3584 CG Utrecht, The Netherlands e-mail: V.Brinks@uu.nl Pharm Res (2013) 30:1729–1734 DOI 10.1007/s11095-013-1067-7