Characterization of the Self-Association of Human Interferon-α 2b, Albinterferon-α 2b, and Pegasys YIMING LI, 1 WALTER F. STAFFORD, 2 MARK HESSELBERG, 1 DAVID HAYES, 2 ZHUCHUN WU, 1 MICHAEL BYRNE 1 1 Human Genome Sciences, Inc., Rockville, Maryland 20850 2 Boston Biomedical Research Institute, Watertown, Massachusetts 02472 Received 17 May 2011; revised 8 August 2011; accepted 16 August 2011 Published online 4 October 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.22751 ABSTRACT: The self-association of human interferon-"2b (hIFN-"2b), albinterferon-"2b (a recombinant protein with human serum albumin and hIFN-"2b peptides fused together in a single polypeptide chain), and Pegasys (PEGylated hIFN-"2a) was characterized by ana- lytical ultracentrifugation analyses. By examining the apparent sedimentation coefficient dis- tribution profiles of each protein at different concentrations, it was concluded that the above three proteins are self-associating in albinterferon-"2b formulation buffer. By model fitting of sedimentation data using SEDANAL software, the stoichiometry and equilibrium constants of the self-association of these proteins were characterized. The self-association of hIFN-"2b results in the formation of stable dimers, fast-reversible tetramers, octamers, and hexade- camers. In contrast, although both albinterferon-"2b and Pegasys are self-associated, their self-association stoichiometries are significantly different from that of hIFN-"2b. The self- association of albinterferon-"2b results in the formation of reversible dimers and trimers, whereas the self-association of Pegasys gives only reversible dimers. The self-association behaviors of hIFN-"2b and albinterferon-"2b involves attractive electrostatic forces, which can be suppressed to a negligible level in low pH (pH 4.0–4.5) and high salt concentration (400mM NaCl) buffer, allowing quantification of their size variant contents by sedimentation velocity analysis. © 2011 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 101:68–80, 2012 Keywords: analytical ultracentrifugation; biopharmaceuticals characterization; macromolec- ular prodrugs; physicochemical properties; physical characterization; proteins INTRODUCTION Interferons (IFNs) are a family of pleiotropic cy- tokines with antiviral, antiproliferation, antitumor, and immunomodulatory properties. 1 Recombinant human interferon (hIFN)-"2a and hIFN-"2b were the first two IFNs licensed by the US Food and Drug Administration for treatment of hairy cell leukemia. Subsequently, these IFNs were approved for clinical use for a variety of viral and cancer indications. 2 Ow- ing to their rapid clearance from the body, frequent dosing (daily or three times weekly) of these two IFNs over an extended period (6–12 months or more) is necessary for some indications such as hepatitis Correspondence to: Yiming Li (Telephone: +301-398-5254; LiYi@Medimmune.com), Walter F. Stafford (Telephone: +617-658- 7808; Stafford@bbri.org) Yiming Li and David Hayes’ present address is Analytical Bio- chemistry, MedImmune, LLC., Gaithersburg, Maryland 20878. Journal of Pharmaceutical Sciences, Vol. 101, 68–80 (2012) © 2011 Wiley Periodicals, Inc. and the American Pharmacists Association B and C. 3 To prolong the serum half-life, PEGylated forms of these two IFNs were developed. PEGylated hIFN-"2a (Pegasys), manufactured by Hoffmann La Roche, Inc. (Basel, Switzerland), is a covalent con- jugate of hIFN-"2a with a single 40 kDa branched bis-monomethoxy-polyethylene glycol (PEG) chain. 4–6 PEGylated hIFN-"2b (Pegintron), manufactured by Schering Corporation (now Merck & Co.) (Kenilworth, New Jersey), is a covalent conjugate of hIFN-"2b with a linear 12 kDa PEG chain. 7 Both products have a half-life much longer than their unmodi- fied counterparts. 8 A different approach to improve the pharmacokinetic properties of IFNs was adopted by Human Genome Sciences, Inc. This approach is based on the expression of the recombinant “gene” formed by fusing DNA sequences encoding human serum albumin (HSA) and hIFN-"2b. The fusion pro- tein manufactured in this manner is referred to as albinterferon-"2b. The clinical trial data showed that albinterferon-"2b has a half-life significantly longer than PEGylated IFNs. 9–11 68 JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 1, JANUARY 2012