Site-Specific Tryptophan Oxidation Induced by Autocatalytic Reaction of Polysorbate 20 in Protein Formulation

被引：51

作者：

Lam, Xanthe M. ^{[1
]}

Lai, William G. ^{[1
]}

Chan, Edwin K. ^{[1
]}

Ling, Victor ^{[2
]}

Hsu, Chung C. ^{[1
]}

机构：

[1] Genentech Inc, Dept Pharmaceut & Proc Dev, San Francisco, CA 94080 USA

[2] Genentech Inc, Dept Prot Analyt Chem, San Francisco, CA 94080 USA

来源：

PHARMACEUTICAL RESEARCH | 2011年 / 28卷 / 10期

关键词：

autocatalytic reaction kinetics; free radicals; peroxides; polysorbate; 20; tryptophan oxidation; HUMAN GROWTH-HORMONE; METHIONINE RESIDUES; MONOCLONAL-ANTIBODY; CATALYZED OXIDATION; IDENTIFICATION; ANTIOXIDANTS; DECOMPOSITION; AUTOXIDATION; BINDING;

D O I：

10.1007/s11095-011-0482-x

中图分类号：

O6 [化学];

学科分类号：

0703 ;

摘要：

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. 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. PS20 with increased peroxides level led to degradation of Fab stored at 30A degrees 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. 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.

引用

页码：2543 / 2555

页数：13

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