Linear and Branched Bicin Linkers for Releasable PEGylation of Macromolecules: Controlled Release in Vivo and in Vitro from Mono- and Multi-PEGylated Proteins Hong Zhao, Karen Yang, Anthony Martinez, Amartya Basu, Ramesh Chintala, Hsien-Ching Liu, Ahsen Janjua, Maoliang Wang, and David Filpula* Enzon Pharmaceuticals, Inc., 20 Kingsbridge Road, Piscataway, New Jersey 08854. Received September 6, 2005; Revised Manuscript Received January 12, 2006 The utility of PEGylation for improving therapeutic protein pharmacology would be substantially expanded if the authentic protein drugs could be regenerated in vivo. Diminution of kinetic constants of both enzymes and protein ligands are commonly encountered following permanent bioconjugation with poly(ethylene glycol) polymers. In further development of releasable linker technology, we investigated an amino PEG anchimeric prodrug system, based on either the linear or branched bicin3 (BCN3) linkage, one promising representative of several aliphatic ester structures synthesized from N-modifed bis-2-hydroxyethylglycinamide (bicin). Protein models included an enzyme, lysozyme, and a receptor ligand, interferon-beta-1b, for preparation of linear or branched mono- and multi-PEGylated conjugates as inactive PEG-BCN3 prodrugs. The kinetics of protein release, both in plasma (in vitro) and in mice (in vivo), correlated with the number of PEG attachments, and the plasma half-lives of PEG release spanned a duration of hours to days within the therapeutically relevant window. Capillary electrophoresis, SDS-PAGE, mass determination, and enzymatic and antiviral activity determinations demonstrated regeneration of equivalent native proteins from the inactive PEG-BCN3 conjugates. Pharmacokinetic analysis of the PEGylated interferon-beta-1b administered subcutaneously in mice demonstrated an over 20-fold expansion of the area under the curve exposure of bioactive protein when compared to native protein. INTRODUCTION Bioconjugation of proteins with poly(ethylene glycol) (PEG 1 ) via permanent linkages, the locus classicus of PEGylation science, is a mature technology employed in at least six PEGylated protein therapeutics which are now on the market (1, 2). A rapid clearance of proteins following parenteral administration may transpire from renal filtration, antibody or cellular recognition, or physical degradation. PEG attachments to proteins have significant benefits in prolonging the in vivo activity of proteins (3-6). A recent trend in PEGylation technology attempts to optimally design the composite biocon- jugate using both protein engineering and improved chemical linkages (7-10). Site-specific attachments of PEG polymer strands may afford better retention of bioactivity, while abridg- ing the heterogeneity of the conjugate compounds. However, even well designed bioconjugates may exhibit imperfect phar- macological properties, owing to an altered structure or function of the bioconjugate when compared to the native protein molecule, which itself may have intrinsic deficiencies when examined in therapeutic applications. Therefore, recent research has examined the potential development of releasable PEGy- lation, wherein covalent attachments of strands of PEG polymers are shed via a controlled release mechanism derived from a degradable linkage at the attachment site on the protein surface (11-19). Releasable PEGylation may offer the greatest benefits to small molecules which are inactivated by the bulky PEG partner. Examples are peptides, oligonucleotides, and analogues of protein domains. In addition, protein therapeutics, such as toxins and signal transduction domains, requiring intracellular entry and processing might also employ a releasable PEG format. Releasable PEGs provide the option of extensive surface modification of highly immunogenic or antigenic proteins with gradual release of the active compound. Finally, it will be instructional to examine the side-by-side in vivo efficacy performance of cytokine or growth factor therapeutics that already demonstrated superior performance as nonreleasable PEGylated derivatives. Two attractive features of rPEGylation that might justify this reinvestigation are (1) the potential for improved manufacturing yields by extensive rPEGylation of the therapeutic, which allows nearly complete product recovery, and (2) the capacity of rPEGylated therapeutics to release the authentic and fully active drug. One prodrug strategy for releasable PEGylation employs the rapid hydrolysis of bis-N-2-hydroxyethylglycinamide (bicin) whereby an anchimeric assisted hydrolysis facilitates the release of amine-conjugated molecules (20). This is a promising linker design that is wholly aliphatic and compatible with amino group derivatization, a common linkage site for protein conjugation. By the use of R-substituents on the ester moiety between the bicin group and the PEG polymer, the half-life of the prodrugs can be adjusted for various applications. However, this PEG- protein conjugate (20, Scheme 1) is a double prodrug, and the stepwise release of PEG from the branched PEG leads initially to a monosubstituted linear PEGylated species. The branched PEG design, featuring more PEG mass per attachment, may be preferable for optimal protection of some therapeutics; however, the two-step releasing mechanism (Scheme 2) may result in excessively complex pharmacokinetic parameters for the re- leased protein. Therefore it is desirable to design a bis-N-2- * To whom correspondence should be addressed. Phone: (732) 980- 4941, Fax: 732-885-2950, E-mail: david.filpula@enzon.com. 1 Abbreviations: PEG, poly(ethylene glycol); BCN3, bicin3; MW, molecular weight; interferon-beta-1b, IFN--1b; rU-PEG, releasable branched (umbrella) linker with two attached PEG polymers; U, branched (umbrella) linker with two attached PEG polymers; rPEGy- lation, releasable PEGylation; CE, capillary electrophoresis; SC, succinimidyl carbonate; SPA, succinimidyl propionate. 341 Bioconjugate Chem. 2006, 17, 341-351 10.1021/bc050270c CCC: $33.50 © 2006 American Chemical Society Published on Web 02/17/2006