Evaluation of the Immuno-Stimulatory Potential of Stopper Extractables and Leachables by Using Dendritic Cells as Readout ROBERT MUELLER, 1 ANETTE KARLE, 2 ANNE VOGT, 2 HARALD KROPSHOFER, 2 ALFRED ROSS, 3 KARSTEN MAEDER, 4 HANNS-CHRISTIAN MAHLER 1 1 F. Hoffmann-La Roche Ltd, Formulation R&D Biologics, Pharmaceutical and Analytical R&D, Basel, Switzerland 2 F. Hoffmann-La Roche Ltd, Immunosafety Operations, Non-Clinical Safety, Basel, Switzerland 3 F. Hoffmann-La Roche Ltd, Discovery Technologies, Basel, Switzerland 4 Institute of Pharmaceutical Technology & Biopharmacy, Martin Luther University Halle-Wittenberg, Halle/Saale, Germany Received 26 May 2008; revised 7 November 2008; accepted 20 November 2008 Published online 18 February 2009 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21672 ABSTRACT: Recombinant protein pharmaceuticals may bear some risks and undesir- able side effects, such as the appearance of immunogenic reactions. The increased incidence of antibody-mediated pure red cell aplasia (PRCA) outside the United States after administration of a human serum albumin (HSA)-free EPREX 1 (recombinant human erythropoietin alpha) formulation was explained with the generation of rubber stopper related leachables, possibly acting as immunogenic adjuvants. In our study, we have investigated the potential of extractable and leachable preparations of three different pharmaceutical relevant stoppers to generate a ‘‘danger signal’’ in a dendritic cell assay. Furthermore, the investigated extractable and leachable preparations were characterized by NMR and a micelle-based polysorbate quantification method. In summary, we could demonstrate that stopper extractables, either generated by extrac- tion or by leaching conditions, were not acting as danger signals for dendritic cells. Instead we identified degradation products of polysorbate 80, oleic acid and follow-up products, occur only under very accelerated conditions (1008C for 4 days) as a potential stimulator for these immune cells. As this degradation did not occur at real-time, the authors however do not consider their finding to be linked to any direct safety implica- tions of polysorbate-containing formulations in clinical practice. ß 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:3548–3561, 2009 Keywords: biotechnology; injectables; formulation; excipients; surfactants; micelle; NMR spectroscopy; immunology INTRODUCTION Recombinant protein pharmaceuticals are widely used for the treatment of a broad range of severe diseases. 1 However, besides the progress in developing active and safe therapeutic proteins, protein-based therapies also may bear some risks and undesirable side effects. One of the issues related to protein pharmaceuticals is the appear- ance of immunogenic reactions to the therapeutic protein. According to the original immunological defini- tion, immunogenicity describes the property of a molecule to provoke an adaptive immune Correspondence to: Hanns-Christian Mahler (Telephone: 41-61-68-83174; Fax: 41-61-68-88689; E-mail: hanns-christian.mahler@roche.com) Journal of Pharmaceutical Sciences, Vol. 98, 3548–3561 (2009) ß 2009 Wiley-Liss, Inc. and the American Pharmacists Association 3548 JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 10, OCTOBER 2009