caa a22 4500 445313056 CHVBK 20180317142636.0 cr unu---uuuuu 170323e20111001xx s 000 0 eng 10.1007/s11095-011-0482-x doi (NATIONALLICENCE)springer-10.1007/s11095-011-0482-x Site-Specific Tryptophan Oxidation Induced by Autocatalytic Reaction of Polysorbate 20 in Protein Formulation [Elektronische Daten] [Xanthe Lam, William Lai, Edwin Chan, Victor Ling, Chung Hsu] ABSTRACT: Purpose: Tryptophan (Trp) oxidation leading to atypical degradation of a protein (Fab) formulated with polysorbate 20 (PS20) was investigated. Such atypical Trp oxidation was discussed in relation to a kinetic model that involves initiation of oxidizing free radical through an autocatalytic reaction. Methods: Ion-exchange chromatography and peptide mapping were used to determine Trp oxidation. Peroxides in PS20 and free radicals in Fab samples were detected by fluorometric assay and electron paramagnetic resonance (EPR), respectively. Results: PS20 with increased peroxides level led to degradation of Fab stored at 30°C. Degradation was characterized as Trp50 oxidation, which was not observed in a Fab variant where His31 was replaced. EPR peaks related to known spin adducts of 5,5 dimethylpyrroline N-oxide were detected in Fab exhibiting Trp oxidation, indicating free radicals were present. Trp oxidation of Fab observed in several drug product lots with different degradation rates fits an autocatalytic reaction model that involves free radicals. EDTA, catalase, and free tryptophan prevented oxidation. Conclusions: A metal-binding amino acid, His31, was responsible for Trp50 oxidation of Fab induced by peroxides in PS20 present in the protein formulation. Oxidation was induced by autocatalytic degradation of PS20 and could be inhibited by antioxidants. Springer Science+Business Media, LLC, 2011 autocatalytic reaction kinetics nationallicence free radicals nationallicence peroxides nationallicence polysorbate 20 nationallicence tryptophan oxidation nationallicence Lam Xanthe Department of Pharmaceutical and Processing Development, Genentech, Inc., 1 DNA Way, 94080, South San Francisco, California, USA aut Lai William Department of Pharmaceutical and Processing Development, Genentech, Inc., 1 DNA Way, 94080, South San Francisco, California, USA aut Chan Edwin Department of Pharmaceutical and Processing Development, Genentech, Inc., 1 DNA Way, 94080, South San Francisco, California, USA aut Ling Victor Department of Protein Analytical Chemistry, Genentech, Inc., 1 DNA Way, 94080, South San Francisco, California, USA aut Hsu Chung Department of Pharmaceutical and Processing Development, Genentech, Inc., 1 DNA Way, 94080, South San Francisco, California, USA aut Pharmaceutical Research Springer US; http://www.springer-ny.com 28/10(2011-10-01), 2543-2555 0724-8741 28:10<2543 2011 28 11095 https://doi.org/10.1007/s11095-011-0482-x text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s11095-011-0482-x text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Lam Xanthe Department of Pharmaceutical and Processing Development, Genentech, Inc., 1 DNA Way, 94080, South San Francisco, California, USA aut NATIONALLICENCE 700 1- Lai William Department of Pharmaceutical and Processing Development, Genentech, Inc., 1 DNA Way, 94080, South San Francisco, California, USA aut NATIONALLICENCE 700 1- Chan Edwin Department of Pharmaceutical and Processing Development, Genentech, Inc., 1 DNA Way, 94080, South San Francisco, California, USA aut NATIONALLICENCE 700 1- Ling Victor Department of Protein Analytical Chemistry, Genentech, Inc., 1 DNA Way, 94080, South San Francisco, California, USA aut NATIONALLICENCE 700 1- Hsu Chung Department of Pharmaceutical and Processing Development, Genentech, Inc., 1 DNA Way, 94080, South San Francisco, California, USA aut NATIONALLICENCE 773 0- Pharmaceutical Research Springer US; http://www.springer-ny.com 28/10(2011-10-01), 2543-2555 0724-8741 28:10<2543 2011 28 11095 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer