Sensitivity of PEGylated Interferon Detection by Anti-Polyethylene Glycol (PEG) Antibodies Depends on PEG Length Ta-Chun Cheng, † Kuo-Hsiang Chuang, ‡ Michael Chen, § Hsin-Ell Wang, ∥ Shey-Cherng Tzou, ⊥ Yu-Cheng Su, #,¶ Chih-Hung Chuang, □ Chien-Han Kao, † Bing-Mae Chen, ¶ Long-Sen Chang, ○ Steve R. Roffler,* ,¶ and Tian-Lu Cheng* ,○,△ † Graduate Institute of Medicine, § Department of Medical Laboratory Science and Biotechnology, and △ Department of Biomedical Science and Environmental Biology, Kaohsiung Medical University, Kaohsiung, Taiwan ‡ Graduate Institute of Pharmacognosy, Taipei Medical University, Taipei, Taiwan ∥ Department of Biomedical Imaging and Radiological Sciences and # Institute of Microbiology and Immunology, National Yang-Ming University, Taipei, Taiwan ⊥ Department of Biological Science and Technology, National Chiao Tung University, Hsin-Chu, Taiwan ¶ Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan □ Institutes of Basic Medical Sciences, National Cheng Kung University, Tainan, Taiwan ○ Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan ABSTRACT: Attachment of poly(ethylene glycol) to pro- teins can mask immune epitopes to increase serum half-life, reduce immunogenicity, and enhance in vivo biological efficacy. However, PEGylation mediated epitope-masking may also limit sensitivity and accuracy of traditional ELISA. We previously described an anti-PEG-based sandwich ELISA for universal assay of PEGylated molecules. Here, we compared the quantitative assessment of PEGylated interfer- ons by anti-PEG and traditional anti-interferon sandwich ELISA. The detection limits for PEG-Intron (12k-PEG) and Pegasys (40k-PEG) were 1.9 and 0.03 ng/mL for anti-PEG ELISA compared to 0.18 and 0.42 ng/mL for traditional anti- interferon sandwich ELISA. These results indicate that the anti-PEG sandwich ELISA was insensitive to PEGylation mediated epitope-masking and the sensitivity increased in proportion to the length of PEG. By contrast, PEG-masking interfered with detection by traditional anti-interferon sandwich ELISA. Human and mouse serum did not affect the sensitivity of anti-PEG ELISA but impeded traditional anti-interferon sandwich ELISA. The anti-PEG sandwich ELISA was comparable to anti-interferon sandwich ELISA and radioassay of 131 I-Pegasys in pharmacokinetic studies in mice. The anti-PEG sandwich ELISA provides a sensitive, accurate, and convenient quantitative measurement of PEGylated protein drugs. ■ INTRODUCTION Polyethylene glycol (PEG) is a highly water-soluble and nontoxic polymer that is approved by the Food and Drug Administration (FDA) for human use. 1 Covalent linkage of PEG to proteins can shield binding sites, minimize proteolytic cleavage, and mask immunogenic sites, thereby prolonging the circulation time in the human body, improving therapeutic efficacy, and reducing the injection frequency to enhance patients’ quality of life. 2−5 Based on these advantages, PEGylation has become one of the most useful pharmaceutical technologies to create protein drugs with markedly improved therapeutic properties compared with their unconjugated counter parts. 6,7 For example, the clinical treatment of chronic hepatitis B and C with interferon-α-2a (Roferon-A) has been limited by the short half-life of IFN-α in patients (less than 12 h), resulting in the need to administer repeated injections at least three times a week to achieve the desired therapeutic benefits. PEGylated interferons, including PEG-Intron (linear PEG 12K -interferon-α-2b) and Pegasys (branched PEG 40K - interferon-α-2a), display dramatically increased half-lives, allowing decreased dosing to once per week. 8 How to efficiently assess the pharmacokinetics of PEGylated drugs is an important issue with the ever increasing number of such compounds under development. Measurement of drug concentrations in serum samples is important for clinical drug development and assessment of drug Received: November 21, 2012 Revised: June 16, 2013 Published: July 9, 2013 Technical Note pubs.acs.org/bc © 2013 American Chemical Society 1408 dx.doi.org/10.1021/bc3006144 | Bioconjugate Chem. 2013, 24, 1408−1413