Selective conjugation of poly(2-ethyl 2-oxazoline) to granulocyte colony stimulating factor Anna Mero a, ⁎, Zhihao Fang b , Gianfranco Pasut a , Francesco M. Veronese a , Tacey X. Viegas b a Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Via F. Marzolo 5, 35131 Padua, Italy b Serina Therapeutics, Inc., 601 Genome Way, Huntsville, AL 35806, USA abstract article info Article history: Received 12 December 2011 Accepted 26 February 2012 Available online 3 March 2012 Keywords: Polymer therapeutics Polyoxazoline Protein pharmaceuticals Granulocyte colony stimulating factor Transglutaminase Reductive alkylation Poly(2-ethyl 2-oxazoline) (PEOZ) is a water-soluble, stable and biocompatible polymer that was prepared in a linear form for the conjugation of protein biomolecules. Polymers of molecular weights ranging from 5 to 20 kDa, with an aldehyde or an amine functional terminal group, were synthesized with narrow polydisper- sities. To assess the suitability of the polymer for therapeutic application, granulocyte colony stimulating fac- tor (G-CSF) was used as a model protein for PEOZ conjugation. Two coupling strategies were employed, namely the chemical N-terminal reductive amination and the enzymatic transglutaminase (TGase) mediated glutamine conjugation. The secondary structure of the protein, measured by circular dichroism, was main- tained upon PEOZylation and the stability of conjugates toward aggregation at 37 °C was improved compared to G-CSF. The potency of PEOZ-G-CSF mono-conjugates was tested in vitro by cell proliferation assays and in vivo by studying the effects on white blood cell and neutrophil count increases in normal rats. The results have shown that PEOZ is suitable for protein conjugation by both chemical and enzymatic methods and that the conjugates of G-CSF retained high biological activity, both in vitro and in vivo. © 2012 Elsevier B.V. All rights reserved. 1. Introduction The biopharmaceutical drug portfolio contains a significant num- ber of naturally occurring and recombinant proteins. These biomol- ecules have been developed as drugs to treat several diseases such as neutropenia, anemia, hepatitis, hemophilia, cancer and Crohn's disease. However, protein drugs might suffer from several limita- tions, such as short blood circulation with poor in vivo residence time, proteolysis, immunogenicity, chemical and physical instability. Protein modification with biocompatible polymer is a well-known method to improve the pharmacological properties and the stability of such drugs. The polymer conjugation reduces proteolytic diges- tion and slows the rate of renal glomerular filtration by providing a camouflage around the protein and producing an increase of its hydrodynamic size. This shield also protects the drug from possible protein antigen-antibody like reactions. So far, PEG is the most used polymer in this field and has achieved wide success in protein drug development as demonstrated by the several approved products [1,2]. It has been covalently attached to many proteins among them granulocyte colony stimulating factor (G-CSF), erythropoietin (EPO), interferon α (IFN), and human growth hormone (hGH) [3]. PEG is biocompatible, non-toxic, safe, widely used as excipient in sev- eral drug formulation, body-care products and food. Nevertheless, some recent reports have shown the presence of specific antibodies against PEG in the serum of patients treated with PEG-asparaginase [4] and PEG-uricase [5] that yielded a loss of therapeutic efficacy. However, it has to be highlighted that in these cases the conjugated proteins were of heterologous origin and very immunogenic and to date it is unclear to what extent the anti-PEG response is related to the nature of the conjugated protein. Another controversy is related to the possible kidney cell vacuolization observed in ani- mals following repeated administrations of PEG conjugates, as demonstrated with TNF-α binding protein, leptin [6], and hemo- globin [7,8]. Also in this case, it is important to note that this vacuolation was not correlated with any renal dysfunction and was reversible after 2 months. Furthermore it has been reported only with certain conjugates administered at high doses, and not with PEG alone. Another constrain for the use of PEG in protein conjugation is the high number of patents issued on claims of composition, methods and application that can hamper the ability to introduce new PEGylated therapies into the clinic. On these bases, companies and research groups have gone to seek and test Journal of Controlled Release 159 (2012) 353–361 Abbreviations: POZ, poly(alkyl-2-oxazoline); PEOZ, poly(2-ethyl 2-oxazoline); PEG, poly(ethylene glycol); HPLC, high performance liquid chromatography; RP, reverse- phase; SEC, size exclusion chromatography; IEC, ionic exchange chromatography; MALDI-TOF, matrix-assisted laser desorption ionization time of flight, G-CSF, human granulocyte colony stimulating factor; TGase, transglutaminase. ⁎ Corresponding author at: Dept. of Pharmaceutical Sciences, University of Padova, Via F. Marzolo 5, 35131, Padova, Italy. Tel.: +39 049 8275693; fax: +39 049 8275366. E-mail address: anna.mero@unipd.it (A. Mero). 0168-3659/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2012.02.025 Contents lists available at SciVerse ScienceDirect Journal of Controlled Release journal homepage: www.elsevier.com/locate/jconrel