title: Evaluation of the immuno-stimulatory potential of stopper extractables and leachables by using dendritic cells as readout.
abstract: Recombinant protein pharmaceuticals may bear some risks and undesirable 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 (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 extraction 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 (100 degrees C 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 implications of polysorbate-containing formulations in clinical practice.
authors.affiliation: F. Hoffmann-La Roche Ltd, Formulation R&D Biologics, Pharmaceutical and Analytical R&D, Basel, Switzerland.
authors.forename: Robert
authors.initials: R
authors.lastname: Mueller
authors.forename: Anette
authors.initials: A
authors.lastname: Karle
authors.forename: Anne
authors.initials: A
authors.lastname: Vogt
authors.forename: Harald
authors.initials: H
authors.lastname: Kropshofer
authors.forename: Alfred
authors.initials: A
authors.lastname: Ross
authors.forename: Karsten
authors.initials: K
authors.lastname: Maeder
authors.forename: Hanns-Christian
authors.initials: HC
authors.lastname: Mahler
mesh_terms: D000276:Adjuvants, Immunologic / Q000737:chemistry* / Q000494:pharmacology*; D000465:Algorithms; D051940:B7-2 Antigen / Q000032:analysis; D002626:Chemistry, Pharmaceutical; D002851:Chromatography, High Pressure Liquid; D003713:Dendritic Cells / Q000187:drug effects*; D004340:Drug Contamination; D004349:Drug Packaging; D000068817:Epoetin Alfa; D004921:Erythropoietin / Q000032:analysis; D005079:Excipients / Q000032:analysis; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008823:Micelles; D009000:Monocytes / Q000187:drug effects; D019301:Oleic Acid / Q000032:analysis; D011136:Polysorbates / Q000032:analysis; D011994:Recombinant Proteins; D012408:Rubber / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet; D015854:Up-Regulation / Q000187:drug effects
chemical_list: D000276:Adjuvants, Immunologic; D051940:B7-2 Antigen; D005079:Excipients; D008823:Micelles; D011136:Polysorbates; D011994:Recombinant Proteins; D004921:Erythropoietin; D019301:Oleic Acid; D000068817:Epoetin Alfa; D012408:Rubber